Method for diagnosing inflammatory bowel disease

ABSTRACT

The present invention relates to a method for identifying a compound capable of modulating the action of the DLG5 protein which method comprises subjecting one or more test compounds to a screen comprising a polypeptide containing the amino acid sequence shown in SEQ ID NO: 2, or a homologue thereof or a fragment of either. Such compounds may be useful in the treatment of inflammatory bowel disease. The present invention also relates to a method for diagnosing inflammatory bowel disease by detecting variant DLG5.

TECHNICAL FIELD

The inventors have discovered a human gene linked to susceptibility toinflammatory bowel disease (IBD) using linkage and association analysis.The present invention therefore relates to diagnostic techniques for thedetection of IBD, and for determining a patient's susceptibility todevelop IBD by detecting all or part of this gene, its precursors orproducts (e.g. mRNA, cDNA, genomic DNA, or protein). The invention isalso directed to methods for identifying modulators of IBD, whichmodulators, such as chemical compounds, antisense molecules andantibodies modulate the gene identified.

BACKGROUND TO INVENTION

Inflammatory bowel disease (IBD) is characterised by a chronic relapsingintestinal inflammation of the gastrointestinal tract. It affects ˜1/1,000 individuals in Western countries with the median age of onset inearly adulthood. To date, the etiology of this disease is unknown. Basedon clinical and histopathological features, IBD is categorised into twomain subtypes, Crohn's disease (CD) (On Line Mendelian Inheritance inMan—a database produced by Johns Hopkins University available at NCBI,OMIM 266600) and ulcerative colitis (UC) (OMIM 191390). Although thecause of IBD is unknown, both familial clustering of the disease andincreased concordance in monozygotic twins shows a strong geneticsusceptibility. Estimates of sibling risk (λs) show a range of 10-50,suggesting that genetic factors play a significant role in thepredisposition to IBD. In the present context the term IBD is intendedto include IBD, as well as Crohn's disease and ulcerative colitis.

Previous genome wide linkage analyses have identified a number ofsusceptibility locus for IBD, e.g. IBD1 (OMIM 266600) (Hugot et al.,Nature. 379:821-823, 1996; Brant et al., Gastroenterlogy 115:1056-1061,1998; Curran et al., Gastroenterology 115: 1066-1071, 1998; and, Hampeet al., Am. J. Hum. Genet. 64:808-816, 1999a), IBD 2 (OMIM 601458)(Duerr et al., Am. J. Hum. Genet 63:95-100, 1988; and, Parkes et al.,Am. J. Hum. Genet. 67:1605-1610, 2000), IBD3 (OMIM 604519) (Hampe etal., Am. J. Hum. Genet. 65:1647-1655, 1999b), IBD7 (OMIM 605225) (Cho etal., Proc. Nat. Acad. Sci. 95:7502-7507; and, Cho et al., Hum. Molec.Genet. 9:1425-1432, 2000).

There is therefore a desire to identify genes with a significantassociation to the development of IBD. This may enable the developmentof novel therapies for IBD by screening for compounds and otherentities, such as antibodies, which modulate the activity of theproteins encoded by the associated genes. Knowledge of the sequence ofthe associated genes may also enable the development of novel antigenemethods to modulate the expression of the associated gene and may alsoenable the development of novel gene therapy techniques to treat IBD.The discovery of associated genes may also assist in developing novelmethods for diagnosing IBD via (i) analysis of the pattern of genotypesof associated single nucleotide polymorphisms (SNPs), (ii) measuring thelevels of the transcribed mRNA present in affected tissue or (iii)measuring the levels of the protein in affected tissue. It is possiblethat the diagnosis of IBD, or the prediction of predisposition to IBD,by these methods may be achieved in patients who do not yet display theclassical symptoms of the disease. Such determination of susceptibilityto IBD or the early detection of disease development may lead to earlierclinical intervention than is currently possible and may lead to moreeffective treatment of the disease.

The present invention is based on our discovery of an association withIBD for a single gene termed dlg5 located on chromosome 10q22.3.

As used herein, the gene is referred to as the dlg1 gene. Specifically,the cDNA sequence is shown in SEQ ID No: 1. Encoded protein is shown inSEQ ID No: 2 and is referred to as DLG5. A C-terminally truncated cDNAsequence is shown in SEQ ID NO: 189 and its encoded protein is shown inSEQ ID NO: 190.

DLG5 belongs to the so-called MAGUK family of proteins (MembraneAssociated Guanylate Kinases, reviewed in Dimitratos et al. BioEssays21:912-921, 1999). Proteins of this family contain several distinctprotein motifs including a guanylate kinase domain, one or several PDZdomains (Postsynaptic density 95, Discs large, Zona occludens-1 domain)and a SH3 domain (src homology domain 3). PDZ domains and SH3 domainshave been shown to mediate protein-protein interactions. In severalcases where PDZ domain interactions have been characterised they havebeen shown to interact with short C-terminal sequences of membraneproteins (Kreienkamp, Curr. Opin. Pharm. 2:581-586, 2002). SH3 domainshave been found to interact with proline rich surface regions of targetproteins. Since the guanylate kinase domain for some MAGUK proteins hasbeen shown to mediate protein-protein interactions while it lacks kinaseactivity, it is generally believed that the main function also for thisdomain is to mediate protein-protein interactions. Therefore, MAGUKproteins are considered as scaffold proteins, orchestrating signallingmolecules to specific membrane locations. Besides establishment of cellpolarity of epithelia, MAGUK proteins have also been implicated inestablishment of postsynaptic compartments in neurons (Kreienkamp, Curr.Opin. Pharm. 2:581-586, 2002). For example, an interaction between a PDZdomain of the MAGUK protein hDLG1/PSD-95 and the intracellular tail ofthe NMDA receptor has been identified. Since it has been identified thatthe C-termini of some 50 intracellular and membrane proteins have highaffinity for the PDZ domains of hDLG-1/PSD-95, it has been hypothesisedthat such clustering of scaffold proteins to multiple membrane receptorsis responsible for localisation of neuroreceptors at postsynaptic sites(Kreienkamp, Curr. Opin. Pharm, 2:581-586, 2002).

Until recently, little has been known about the function of DLG5.Partial EST sequences for dlg5 were identified from a database search byits similarity to other MAGUK proteins (Nakamura et al., FEBS 433:63-67,1998). The authors identified a partial cDNA sequence referred to asP-dlg and showed by immunostaining that the protein was expressed inepithelial gland cells of the prostate. It was also shown by atwo-hybrid screen that the DLG5/P-dlg specifically interacted with p55,another MAGUK protein. Northern blot analyses showed variable expressionin multiple tissues (Nakamura et al., FEBS 433:63-67, 1998 and Shah etal., BMC Genomics 3:6 2002). Shah et al. also showed that the human geneconsisting of 32 exons, encoded a full length DLG5 protein of 1809 aminoacids. The DLG5 protein contains 4 PDZ domains followed by an SH3 domainand a C terminal guanylate kinase domain.

It was recently shown that DLG5 could be identified in a two-hybridscreen using vinexin as bait (Wakabayashi et al., JBC, 25th Mar.20032003). Furthermore, the authors showed that vinexin, DLG5 andβ-catenin could form a ternary complex, providing a direct link to theadhesion junction complex in epithelial cells.

In vertebrate gut epithelial cells three types of cell junctions areformed (reviewed in Tsukita et al,. Nature Rev. Mol. Cell. Biol.2:285-293, 2001). Tight junctions are located towards the apical borderof the basolateral side and are considered to function both as a barrierfor the extracellular environment as well as a fence for membranediffusion. Adherence junctions are formed immediately basolateral oftight junctions and their role is less clear than for tight junctions.They are considered to be important for the mechanical strength of cellcontacts, but it is also clear that their regulation has to be preciselycoordinated with tight junctions, for example when immune cells passesthrough the epithelial barrier. The mutual dependence between tightjunctions and adherence junctions are underscored by the findings thatwhile formation of tight junctions does not occur until adherencejunctions are intact, adherence junctions can not form when formation oftight junctions are inhibited by overexpression of a dominant negativemutant of the tight junction MAGUK protein ZO-3 (Wittchen et al., J.Cell. Biol. 151:825-836, 2000; and refs therein). Finally, multipledesmosomes are located along the basolateral sides and are mainlyconsidered to contribute to the mechanical strength of cell contacts.

Many proteins have been shown to be localised to cellular junctions.Membrane proteins, such as for example occludin and claudins, are foundat tight junctions while members of the cadherin family mediatescell-cell contacts at adherence junctions. A large number of proteinsconnect to the cytoplasmatic side of these membrane proteins, linkingthe complexes both to the actin cytoskeleton and to intracellularsignaling. At adherence junctions, the cytoplasmatic part of cadherinbinds β-catenin, thus providing a link between DLG5 and adherencejunctions.

The background data above strongly supports a functional role for DLG5in gut epithelial cell function and integrity. The inventors proposethat protein(s) encoded by the dlg5 gene, which has only now beenidentified as being genetically linked to susceptibility to IBD, aredirectly or indirectly involved in the pathogenesis of gut inflammation.

The inventors have identified 20 unique nucleotide variations within thedlg5 gene, four of these result in codon changes, a further two aredeletions.

SUMMARY OF THE INVENTION

The inventors have identified a gene located on chromosome 10q22.3,termed dlg5, which demonstrates genetic association linkage tosusceptibility to IBD. The gene, mRNA (or cDNA prepared therefrom) andprotein sequences corresponding to such transcript are thereforediagnostic or prognostic markers of IBD, and can be used to designspecific probes, or to generate antibodies, capable of detecting thepresence of nucleotide sequence polymorphims or mutations of the gene ormRNA, or of measuring the levels of the mRNA or encoded protein presentin a test sample, such as a body fluid or cell sample. In addition thegene and protein encoded thereby is a potential target for therapeuticintervention in IBD disease, for instance in the development ofantisense nucleic acid targeted to the mRNA, or transgenic therapies; ormore widely in the identification or development of chemical or hormonaltherapeutic agents. The person skilled in the art is also capable ofdevising screening assays to identify compounds (chemical or biological)that modulate (activate or inhibit) the identified gene or encodedprotein, which compounds may prove useful as therapeutic agents intreating or preventing IBD.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention there is provided a methodfor identifying a compound capable of modulating the action of the DLG5protein which method comprises subjecting one or more test compounds toa screen comprising a polypeptide containing the amino acid sequenceshown in SEQ ID NO: 2, or a homologue thereof or a fragment of either.

The term “fragment” as used herein refers to a subsequence of the fulllength sequence that comprises at least 25, preferably at least 50, morepreferably at least 100 consecutive amino acids of the sequence depictedin SEQ ID NO: 2, preferably the fragment is a polypeptide that is theDLG5 protein with either or both C-terminal and N-terminal truncations,such as the polypeptide depicted in SEQ ID NO: 190.

It is understood that the polypeptide for use in the invention may beboth a fragment and a homologue of the DLG5 protein.

In a preferred embodiment, the screening methods of the invention arecarried out using a polypeptide comprising an amino acid sequence asdepicted in SEQ ID NO: 2, or a sequence possessing, in increasing orderof preference, at least 80%, 85%, 90%, 95%, 97%, 98% and 99% amino acidsequence identity thereto. Such variants are herein referred to as“homologues”.

The sequence identity between two sequences can be determined bypair-wise computer alignment analysis, using programs such as, BestFit,Gap or FrameAlign. The preferred alignment tool is BestFit. In practise,when searching for similar/identical sequences to the query search, fromwithin a sequence database, it is generally necessary to perform aninitial identification of similar sequences using suitable software suchas Blast, Blast2, NCBI Blast2, WashU Blast2, FastA, Fasta3 and PILEUP,and a scoring matrix such as Blosum 62. Such software packages endeavourto closely approximate the “gold-standard” alignment algorithm ofSmith-Waterman. Thus, the selected software/search engine programme foruse in assessing identity/similarity, i.e how two primary polypeptidesequences line up is Smith-Waterman. Identity refers to direct matches,similarity allows for conservative substitutions.

Allelic variants or versions of the DLG5 protein may exist within thehuman population, particularly between distinct ethnic groups. A furtheraspect of the invention involves the selection and use of theappropriate version of the DLG5 protein to be included in screens so asto discover compounds capable of altering the activity of said DLG5version in vivo. The inventors have identified four codon changingnucleotide polymorphisms within one or other exons of dlg5 gene, each ofthese, alone or in combination, would provide numerous allelic variantprotein versions of DLG5 for use in any aspect of the present invention.

Investigators may wish to screen their compounds against the mostprevalent version of the DLG5 protein and also against the less frequentversions of the DLG5 protein in order to detect any differentialpharmacological activity between the various versions of the target. Afurther aspect of the invention is the screening of various ethnic basedpopulations to measure the allele frequencies of the nucleotidepolymorphisms in the dlg5 gene within said populations. This informationmay be of value in estimating the efficacy of new compounds capable ofaltering the activity of DLG5 within these populations and in particularin estimating the proportion of the population which may not respond tothe therapy.

Polymorphism refers to the occurrence of two or more geneticallydetermined alternative alleles or sequences within a population. Apolymorphic marker is the site at which divergence occurs. Preferablymarkers have at least two alleles, each occurring at frequency ofgreater than 1%, and more preferably at least 10%, 15%, 20%, 30% or moreof a selected population.

Single nucleotide polymorphisms (SNP) are generally, as the nameimplies, single nucleotide or point variations that exist in the nucleicacid sequence of some members of a species. Such polymorphism variationwithin the species, is generally regarded to be the result ofspontaneous mutation throughout evolution. The mutated and normalsequences co-exist within the species' population sometimes in a stableor quasi-stable equilibrium. At other times the mutation may confer someadvantage to the species and with time may be incorporated into thegenomes of all or a majority of members of the species.

Some SNPs occur in the protein coding sequences, in which case, one ofthe polymorphic protein forms may possess a different amino acid whichmay give rise to the expression of a variant protein and, potentially, agenetic disease. These changes in function may be mediated by severalmechanisms including, but not limited to, alterations in proteinfolding, alterations in ligand and substrate binding affinity andalterations in membrane binding affinity and may lead to gain ofactivity or loss of activity for the protein in vivo. Such alterationsin the activity of the protein in vivo may be of clinical significancein the development of IBD. Alteration to the amino acid sequence of theprotein may also affect the efficacy of drug therapy for IBD by alteringthe specificity between protein and compounds selected by screening tomodulate its activity. Thus compounds selected by screening may havedifferent efficacies in modulating the activity of protein in differentindividuals according to the versions of the gene that they carry. Inparticular an individual who is homozygous for a less common variant ofthe gene may not respond well to a therapy developed by screeningcompounds against the dominant variant.

The screening methods according to the invention may be carried outusing conventional procedures, for example by bringing the test compoundor compounds to be screened and an appropriate substrate into contactwith the polypeptide, or a cell capable of producing it, or a cellmembrane preparation thereof, and determining affinity for thepolypeptide in accordance with standard techniques.

Any compound identified in this way may prove useful in the treatment ofIBD in humans and/or other animals. The invention thus extends to acompound selected through its ability to regulate the activity of theDLG5 protein in vivo as primarily determined in a screening assayutilising the polypeptide containing an amino acid sequence shown in SEQID NO: 2, or a homologue or fragment thereof, or a gene coding therefore(such as that disclosed in SEQ ID NO: 1) for use in the treatment of adisease in which the over- or under-activity or unregulated activity ofthe protein is implicated.

According to a further aspect of the invention there is provided ascreening assay or method for identifying potential anti-IBD therapeuticcompounds comprising contacting an assay system capable of detecting theeffect of a test compound against expression level of DLG5, with a testcompound and assessing the change in expression level of DLG5.

Compounds that modulate the expression of DNA or RNA of DLG5polypeptides may be detected by a variety of assay systems. A suitableassay system may be a simple “yes/no” assay to determine whether thereis a change in expression of a reporter gene, such asbeta-galactosidase, luciferase, green fluorescent protein or othersknown to the person skilled in the art (reviewed by Naylor, Biochem.Pharmacol. 58:749-57, 1999). The assay system may be made quantitativeby comparing the expression or function of a test sample with the levelsof expression or function in a standard sample. Systems in whichtranscription factors are used to stimulate a positive output, such astranscription of a reporter gene, are generally referred to as“one-hybrid systems” (Wang, M. M. and Reed, R. R. (1993) Nature364:121-126). Using a transcription factor to stimulate a negativeoutput (growth inhibition) may thus be referred to as a “reverseone-hybrid system” (Vidal et al, 1996, supra). Therefore, in anembodiment of the present invention, a reporter gene is placed under thecontrol of the dlg5 promoter. A suitable dlg5 promoter sequence isdisclosed in SEQ ID NO: 5.

In a further aspect of the invention we provide a cell or cell linecomprising a reporter gene under the control of the dlg5 promoter.

According to another aspect of the present invention there is provided amethod of screening for a compound potentially useful for treatment ofIBD, which comprises assaying the compound for its ability to modulatethe activity or amount of DLG5. Preferably the assay is selected from:

-   (i) measurement of DLG5 activity using a cell line which expresses    the DLG5 polypeptide or using purified DLG5 polypeptide; and-   (ii) measurement of dlg5 transcription or translation in a cell line    expressing the DLG5 polypeptide.

The “DLG5 polypeptide” refers to the DLG5 protein, a homologue thereof,or a fragment of either.

Thus, in a further aspect of the invention, cell cultures expressing theDLG5 polypeptide can be used in a screen for therapeutic agents. Effectsof test compounds may be assayed by changes in mRNA or protein of DLG5.As described below, cells (i.e. mammalian, bacterial, yeast etc.) can beengineered to express the DLG5 polypeptide.

Thus, according to a further aspect of the invention there is provided amethod of testing potential therapeutic agents for the ability tosuppress IBD phenotype comprising contacting a test compound with a cellengineered to express the DLG5 polypeptide; and determining whether saidtest compound modulates expression of the DLG5 polypeptide.

We also provide a method for identifying inhibitors of transcription ofdlg5, which method comprises contacting a potential therapeutic agentwith a cell or cell line as described above and determining inhibitionof dlg5 transcription by the potential therapeutic agent by reference toa lack of or reduction in expression of the reporter gene.

Any convenient test compound or library of test compounds may be used inconjunction with the test assay. Particular test compounds include lowmolecular weight chemical compounds (preferably with a molecular weightless than 1500 daltons) suitable as pharmaceutical or veterinary agentsfor human or animal use, or compounds for non-administered use such ascleaning/sterilising agents or for agricultural use. Test compounds mayalso be biological in nature, such as antibodies.

According to a further aspect of the invention there is provided acompound identified by a screening method as defined herein.

According to another aspect of the present invention there is provideduse of a compound able to modulate the activity or amount of DLG5 in thepreparation of a medicament for the treatment of IBD. Modulation of theamount of DLG5 by a compound may be brought about for example throughaltered gene expression level or message stability. Modulation of theactivity of DLG5 by a compound may also be brought about for examplethrough compound binding to the DLG5 protein. In one embodiment,modulation of DLG5 comprises use of a compound able to reduce theactivity or amount of DLG5. In another embodiment, modulation of DLG5comprises use of a compound able to increase the activity or amount ofDLG5.

It will be appreciated that the term ‘for the treatment of IBD’, andvariations thereon, includes therapeutic and prophylactic (preventative)treatment.

According to another aspect of the present invention there is provided amethod of preparing a pharmaceutical composition which comprises:

-   i) identifying a compound as useful for treatment of IBD according    to a screening method as described herein; and-   ii) mixing the compound or a pharmaceutically acceptable salt    thereof with a pharmaceutically acceptable excipient or diluent.

According to a further aspect of the invention there is provided amethod of treatment of a patient suffering from IBD comprisingadministration to said patient of an effective amount of a compoundidentified according to a screening method of the invention or acomposition prepared by the method described herein.

The compositions of the invention may be in a form suitable for oral use(for example as tablets, lozenges, hard or soft capsules, aqueous oroily suspensions, emulsions, dispersible powders or granules, syrups orelixirs), for topical use (for example as creams, ointments, gels, oraqueous or oily solutions or suspensions), for administration byinhalation (for example as a finely divided powder or a liquid aerosol),for administration by insufflation (for example as a finely dividedpowder) or for parenteral administration (for example as a sterileaqueous or oily solution for intravenous, subcutaneous, intramuscular orintramuscular dosing or as a suppository for rectal dosing).

The compositions of the invention may be obtained by conventionalprocedures using conventional pharmaceutical excipients, well known inthe art. Thus, compositions intended for oral use may contain, forexample, one or more colouring, sweetening, flavouring and/orpreservative agents.

Suitable pharmaceutically acceptable excipients for a tablet formulationinclude, for example, inert diluents such as lactose, sodium carbonate,calcium phosphate or calcium carbonate, granulating and disintegratingagents such as corn starch or algenic acid; binding agents such asstarch; lubricating agents such as magnesium stearate, stearic acid ortalc; preservative agents such as ethyl or propyl p-hydroxybenzoate, andanti-oxidants, such as ascorbic acid. Tablet formulations may beuncoated or coated either to modify their disintegration and thesubsequent absorption of the active ingredient within thegastrointestinal track, or to improve their stability and/or appearance,in either case, using conventional coating agents and procedures wellknown in the art.

Compositions for oral use may be in the form of hard gelatin capsules inwhich the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules in which the active ingredient is mixed with water oran oil such as peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions generally contain the active ingredient in finelypowdered form together with one or more suspending agents, such assodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone,gum tragacanth and gum acacia; dispersing or wetting agents such aslecithin or condensation products of an alkylene oxide with fatty acids(for example polyoxethylene stearate), or condensation products ofethylene oxide with long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives (such as ethyl orpropyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid),colouring agents, flavouring agents, and/or sweetening agents (such assucrose, saccharine or aspartame).

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil (such as arachis oil, olive oil, sesame oil orcoconut oil) or in a mineral oil (such as liquid paraffin). The oilysuspensions may also contain a thickening agent such as beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set outabove, and flavouring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water generally contain the activeingredient together with a dispersing or wetting agent, suspending agentand one or more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients such as sweetening, flavouring and colouringagents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, or a mineral oil, such as for exampleliquid paraffin or a mixture of any of these. Suitable emulsifyingagents may be, for example, naturally-occurring gums such as gum acaciaor gum tragacanth, naturally-occurring phosphatides such as soya bean,lecithin, an esters or partial esters derived from fatty acids andhexitol anhydrides (for example sorbitan monooleate) and condensationproducts of the said partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening, flavouring and preservative agents.

Syrups and elixirs may be formulated with sweetening agents such asglycerol, propylene glycol, sorbitol, aspartame or sucrose, and may alsocontain a demulcent, preservative, flavouring and/or colouring agent.

The pharmaceutical compositions may also be in the form of a sterileinjectable aqueous or oily suspension, which may be formulated accordingto known procedures using one or more of the appropriate dispersing orwetting agents and suspending agents, which have been mentioned above. Asterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally-acceptable diluent or solvent,for example a solution in 1,3-butanediol.

Suppository formulations may be prepared by mixing the active ingredientwith a suitable non-irritating excipient, which is solid at ordinarytemperatures but liquid at the rectal temperature and will thereforemelt in the rectum to release the drug. Suitable excipients include, forexample, cocoa butter and polyethylene glycols.

Topical formulations, such as creams, ointments, gels and aqueous oroily solutions or suspensions, may generally be obtained by formulatingan active ingredient with a conventional, topically acceptable, vehicleor diluent using conventional procedure well known in the art.

Compositions for administration by insufflation may be in the form of afinely divided powder containing particles of average diameter of, forexample, 30μ or much less, the powder itself comprising either activeingredient alone or diluted with one or more physiologically acceptablecarriers such as lactose. The powder for insufflation is thenconveniently retained in a capsule containing, for example, 1 to 50 mgof active ingredient for use with a turbo-inhaler device, such as isused for insufflation of the known agent sodium cromoglycate.

Compositions for administration by inhalation may be in the form of aconventional pressurised aerosol arranged to dispense the activeingredient either as an aerosol containing finely divided solid orliquid droplets. Conventional aerosol propellants such as volatilefluorinated hydrocarbons or hydrocarbons may be used and the aerosoldevice is conveniently arranged to dispense a metered quantity of activeingredient.

For further information on Formulation the reader is referred to Chapter25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch;Chairman of Editorial BIBDrd), Pergamon Press 1990.

The amount of active ingredient that is combined with one or moreexcipients to produce a single dosage form will necessarily varydepending upon the host treated and the particular route ofadministration. For example, a formulation intended for oraladministration to humans will generally contain, for example, from 0.5mg to 2 g of active agent compounded with an appropriate and convenientamount of excipients which may vary from about 5 to about 98 percent byweight of the total composition. Dosage unit forms will generallycontain about 1 mg to about 500 mg of an active ingredient. For furtherinformation on Routes of Administration and Dosage Regimes the reader isreferred to Chapter 25.3 in Volume 5 of Comprehensive MedicinalChemistry (Corwin Hansch; Chairman of Editorial BIBDrd), Pergamon Press1990.

The size of the dose for therapeutic or prophylactic purposes of acompound will naturally vary according to the nature and severity of theconditions, the age and sex of the animal or patient and the route ofadministration, according to well known principles of medicine.

In using a compound for therapeutic or prophylactic purposes it willgenerally be administered so that a daily dose in the range, forexample, 0.5 mg to 75 mg per kg body weight is received, given ifrequired in divided doses. In general lower doses will be administeredwhen a parenteral route is employed. Thus, for example, for intravenousadministration, a dose in the range, for example, 0.5 mg to 30 mg per kgbody weight will generally be used. Similarly, for administration byinhalation, a dose in the range, for example, 0.5 mg to 25 mg per kgbody weight will be used. Oral administration is however preferred.

Having identified that the dlg5 gene is implicated in IBD, this presentsmany molecular diagnostic opportunities. It is known to persons skilledin the art that clinically significant information may be obtained bythe measurement of the levels of nucleic acids, proteins or otheranalytes that occur within biological samples. When nucleic acids,proteins or other analytes occur in polymorphic form then there may alsobe diagnostic utility in by identifying which of the various versions ofsaid polymorphic nucleic acids, proteins or other analytes occur withina sample.

An investigator may wish to measure the levels of DLG5 protein or tomeasure the levels of dlg5 mRNA transcript present in a sample. Aninvestigator may also wish to perform nucleic acid sequence analyses todetect variant nucleotides present within the sample, these analyses maybe performed on either the DNA or RNA fraction of the sample and arewell known to the person skilled in the art. An investigator may alsowish to perform protein sequence analysis either directly, bydegradation based techniques which are well known in the art, orindirectly by molecular recognition techniques including immunoassay, orby techniques based on detecting changes in the physical characteristicsof the protein such as functional or substrate specificity assays oriso-electric focusing.

According to a further aspect of the invention there is provided amethod for diagnosing or prognosing or monitoring IBD, comprisingtesting a biological sample for aberrant levels of DLG5.

The term “aberrant levels” refers to levels that are outside the normalrange. The normal range can be determined by testing many normal tissuesor may be determined from a side by side comparison of the test samplewith the normal or control sample. For the purposes of this application,aberrant expression refers to a 1.5-fold difference or more in level ofnucleic acid in a disease sample compared to control normal. Nucleicacid as used herein refers to both RNA and DNA.

The test biological sample is conveniently a sample of sinovial fluid,blood, buccal scrape, urine or other body fluid or tissue obtained froman individual.

The invention lies in the identification of the gene identified hereinbeing linked to IBD disease prevalence. Accordingly, in part, theinvention is directed to any diagnostic method capable of assessing thedifferential expression level, relative to expression in controltissues, of the dlg5 gene identified herein, either alone or as a panel.In particular, such methods include assessment of mRNA transcript levelsand/or protein levels. The presence of aberrant expression levels of thegene indicating the presence of IBD or an increased likelihood todevelop the disorder.

As noted above, in one embodiment the diagnostic/detection methods ofthe invention are employed to detect the presence of one or more SNPs orsmall insertions, deletions or duplications of DLG5 or dlg5. SuitableSNPs and deletions of DLG5 or dlg5 include those identified in Table 3.

Knowledge of polymorphisms can be of assistance in identifying patientssusceptible to particular diseases and those most suited to therapy withparticular pharmaceutical agents (the latter is often termed“pharmacogenetics”). Pharmacogenetics can also be used in pharmaceuticalresearch to assist the drug selection process. Polymorphisms are used inmapping the human genome and to elucidate the genetic component ofdiseases. The reader is directed to the following references forbackground details on pharmacogenetics and other uses of polymorphismdetection: Linder et al. (1997), Clinical Chemistry, 43:254; Marshall(1997), Nature Biotechnology. 15:1249; International Patent ApplicationWO 97/40462, Spectra Biomedical; and Schafer et al, (1998), NatureBiotechnology. 16:33.

A haplotype is a set of alleles found at linked polymorphic sites (suchas within a gene) on a single (paternal or maternal) chromosome. Ifrecombination within the gene is random, there may be as many as 2^(n)haplotypes, where 2 is the number of alleles at each SNP and n is thenumber of SNPs. One approach to identifying mutations or polymorphismswhich are correlated with clinical response is to carry out anassociation study using all the haplotypes that can be identified in thepopulation of interest. The frequency of each haplotype is limited bythe frequency of its rarest allele, so that nucleotide sequencepolymorphisms with low frequency alleles are particularly useful asmarkers of low frequency haplotypes. As particular mutations orpolymorphisms associated with certain clinical features, such as adverseor abnormal events, are likely to be of low frequency within thepopulation, low frequency nucleotide sequence variations may beparticularly useful in identifying these mutations (for examples see:Linkage disequilibrium at the cystathionine beta synthase (CBS) locusand the association between genetic variation at the CBS locus andplasma levels of homocysteine (De Stefano et al., Ann Hum Genet (1998)62:481-90; and, Keightley et al., Blood (1999) 93:4277-83).

Clinical trials have shown that patient response to treatment withpharmaceuticals is often heterogeneous. Thus there is a need forimproved approaches to pharmaceutical agent design and therapy.

Point mutations in polypeptides will be referred to as follows: naturalamino acid (using 1 or 3 letter nomenclature), position, new amino acid.For (a hypothetical) example “D25K” or “Asp25Lys” means that at position25 an aspartic acid (D) has been changed to lysine (K).

The presence of a particular nucleic acid base at a polymorphismposition will be represented by the base following the polymorphismposition. For (a hypothetical) example, the presence of adenine atposition 300 will be represented as: 300A.

We provide examples of nucleotide polymorphisms, including those thataffect the amino acid sequence of the DLG5 protein. Such amino acidchanging polymorphisms are indicated in Table 3 as “non-synonymous”.

Nucleotide polymorphisms (mutations) in the promoter and UTR regions mayalso affect the transcription and expression of the dlg5 gene leading toeither increased or decreased levels of expression or to unregulatedactivity of the DLG5 protein in vivo. Such alterations in the level ofexpression of the DLG5 protein in vivo may result in a gain or loss offunction, which is of clinical significance. Recently, it has beenreported that even polymorphisms that do not result in an amino acidchange can cause different structural folds of mRNA with potentiallydifferent biological functions (Shen et al., (1999) Proc Natl Acad SciUSA 96:7871-7876).

In one embodiment of the invention the screening methods describedherein utilise a DLG5 protein variant which is transcribed from anucleic acid sequence based on that shown in SEQ ID NO:1 or 6.

Nucleotide polymorphisms within dlg5 or DLG5 may also be used asdiagnostic markers of predisposition to disease. Genotyping nucleotidesequence variants in populations suffering from IBD and in controlpopulations not suffering from IBD but matched for factors including,but not limited to, racial ancestry, country of origin, sex, age andbody mass index may allow investigators to identify increased riskfactors associated with the development of IBD disease according to theinheritance of certain SNP genotypes or haplotypes which are moreprevalent in populations with IBD compared to their incidence in thecorresponding control populations. This may enable screening forindividuals at increased risk of developing IBD by measuring thegenotypes and haplotypes of these nucleotide sequence polymorphismswithin non-symptomatic individuals. We have discovered novel sequencepolymorphisms in the dlg5 gene which may be useful for the diagnosis ofIBD. Public domain nucleotide sequence variations, which may also beuseful for the diagnosis of IBD, or for research into IBD, are alsoidentified herein. Table 3 lists the sequence polymorphisms in dlg5.Those database-derived polymorphisms present in the public domain areannotated as either rs- or tsc-. The other SNPs, annotated DLGe, arebelieved to be identified herein, for the first time. Tsc-stands for theSNP consortium.

A nucleotide sequence variation or polymorhisms could be a singlenucleotide polymorhism, a deletion of one or several nucleotides, aduplication of one or several nucleotides or an insertion of one orseveral nucleotides in the nucleotide sequence of the gene or insequences modulating the expression of the dlg5 gene.

According to one aspect of the present invention there is provided amethod for the diagnosis of a single nucleotide polymorphism associatedwith IBD, which method comprises determining from human nucleic acid,the identity of the nucleotide at position 16 according to one or moreof SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93; and determining thestatus of the human by reference to polymorphism(s) detected. Withrespect to the mutation disclosed in SEQ ID NO: 32 and 33, thenucleotide at position 16 will either be C, or in the allele with the7-base deletion, a G.

The term human includes both a human having or suspected of havinginflammatory bowel disease and an asymptomatic human who may be testedfor predisposition or susceptibility to IBD. At each position the humanmay be homozygous for an allele or the human may be a heterozygote.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 7) is the presence of Gand/or A.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 9) is the presence of Cand/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 11) is the presence of Cand/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 13) is the presence of Gand/or A.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 15) is the presence of Gand/or C.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 17) is the presence of Cand/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 19) is the presence of Aand/or G.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 21) is the presence of Cand/or A.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 23) is the presence of Cand/or A.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 25) is the presence of Gand/or A.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 27) is the presence of Cand/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 29) is the presence of Cand/or G.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 3 1) is the presence ofC and/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 35) is the presence of Gand/or A.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 37) is the presence of Gand/or C.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 39) is the presence of Gand/or A.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 41) is the presence of Cand/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 43) is the presence of Gand/or A.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 45) is the presence of Cand/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 47) is the presence of Cand/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 49) is the presence of Cand/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 51) is the presence of Cand/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 53) is the presence of Gand/or C.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 55) is the presence of Cand/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 57) is the presence of Gand/or C.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 59) is the presence of Cand/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 61) is the presence of Cand/or A.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ED NO: 63) is the presence of Tand/or A.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 65) is the presence of Cand/or G.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 67) is the presence of Gand/or A.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 69) is the presence of Cand/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 71) is the presence of Aand/or (3 (as a result of a single base deletion).

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 73) is the presence of Cand/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 75) is the presence of Cand/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 77) is the presence of Cand/or A.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 79) is the presence of Cand/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 81) is the presence of Cand/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 83) is the presence of Gand/or A.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 85) is the presence of Cand/or T.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 87) is the presence of Aand/or G.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 89) is the presence of Gand/or A.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 91) is the presence of Cand/or G.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which the single nucleotide polymorphismlocated at position 16 (according to SEQ ID NO: 93) is the presence of Gand/or A.

In one embodiment of the invention preferably the method for diagnosisdescribed herein is one in which there is the presence or absence of a7-base deletion located at position 16 (according to SEQ ID NO: 33).

In another aspect of the invention there is provided a method for thediagnosis of IBD or determining susceptibility to develop IBD, whichmethod comprises:

-   (i) obtaining a protein or nucleic acid containing sample from an    individual;-   (ii) detecting the presence or absence of a variant DLG5 on the    basis of the presence of a polymorphic amino acid within the DLG5    protein, or a polymorphic base within the dlg5 gene sequence; and,-   (iii) determining the status of the human by reference to the    presence or absence of a polymorphism in DLG5.

In one embodiment the polymorphic amino acid is located at position 140,231, 624, 1067, 1089 or 1481 according to SEQ ID NO: 2.

In a particular embodiment the polymorphism is selected from the groupconsisting of: Gln140Arg, Ser321Gly, Glu624Gln, Arg1067His, Pro1089Leuand Pro1481Gln according to SEQ ID NO: 2.

In one embodiment the polymorphic amino acid is located at position 30,121, 514, 957, 979 or 1371 according to SEQ ID NO: 190.

In a particular embodiment the polymorphism is selected from the groupconsisting of: Gln30Arg, Ser121Gly, Glu514Gln, Arg957His, Pro979Leu andPro1371Gin according to SEQ ID NO: 190.

The protein or nucleic acid containing test sample may conveniently be asample of blood, bronchoalveolar lavage fluid, sputum, or other bodyfluid or tissue obtained from an individual. It will be appreciated thatthe test sample may equally be a nucleic acid sequence corresponding tothe sequence in the test sample, that is to say that all or a part ofthe region in the sample nucleic acid may firstly be amplified using anyconvenient technique e.g. PCR, before use in the analysis of DLG5variation.

It will be apparent to the person skilled in the art that there are alarge number of analytical procedures which may be used to detect thepresence or absence of variant nucleotides at one or more polymorphicpositions of the invention. In general, the detection of allelicvariation requires a mutation discrimination technique, optionally anamplification reaction and optionally a signal generation system. List 1identifies a number of mutation detection techniques, some based on thepolymerase chain reaction (PCR). These may be used in combination with anumber of signal generation systems, a selection of which is listed inList 2. Further amplification techniques are listed in List 3. Manycurrent methods for the detection of allelic variation are reviewed byNollau et al., Clin. Chem. 43, 1114-1120, 1997; and in standardtextbooks, for example “Laboratory Protocols for Mutation Detection”,Ed. by U. Landegren, Oxford University Press, 1996 and “PCR”, 2^(nd)Edition by Newton & Graham, BIOS Scientific Publishers Limited, 1997.TABLE 1 Abbreviations: ALEX ™ Amplification refractory mutation systemlinear extension APEX Arrayed primer extension ARMS ™ Amplificationrefractory mutation system b-DNA Branched DNA CMC Chemical mismatchcleavage Bp base pair COPS Competitive oligonucleotide priming systemDGGE Denaturing gradient gel electrophoresis FRET Fluorescence resonanceenergy transfer LCR Ligase chain reaction MASDA Multiple allele specificdiagnostic assay NASBA Nucleic acid sequence based amplification OLAOligonucleotide ligation assay PCR Polymerase chain reaction PTT Proteintruncation test RFLP Restriction fragment length polymorphism SDA Stranddisplacement amplification SERRS Surface enhanced raman resonancespectroscopy SNP Single nucleotide polymorphism SSCP Single-strandconformation polymorphism analysis SSR Self sustained replication TGGETemperature gradient gel electrophoresis 3′ UTR 3′ untranslated regionList 1—Mutation Detection Techniques

General: DNA sequencing, Sequencing by hybridisation

Scanning: PTT*, SSCP, DGGE, TGGE, Cleavase, Heteroduplex analysis, CMC,Enzymatic mismatch cleavage

*Note: not useful for detection of promoter polymorphisms.

Hybridisation Based: Solid phase hybridisation: Dot blots, MASDA,Reverse dot blots, Oligonucleotide arrays (DNA Chips)

Solution phase hybridisation: Taqman™—U.S. Pat. No. 5,210,015 & U.S.Pat. No. 5,487,972 (Hoffmann-La Roche), Molecular Beacons—Tyagi et al(1996), Nature Biotechnology, 14, 303; WO 95/13399 (Public Health Inst.,New York)

Extension Based: ARMS™-allele specific amplification (as described inEuropean patent No. EP-B-332435 and U.S. Pat. No. 5,595,890),ALEX™—European Patent No. EP 332435 B1 (Zeneca Limited), COPS—Gibbs etal (1989), Nucleic Acids Research, 17, 2347.

Incorporation Based: Mini-sequencing, APEX

Restriction Enzyme Based: RFLP, Restriction site generating PCR

Ligation Based: OLA

Other: Invader assay, Hybridisation protection assay

List 2—Signal Generation or Detection Systems

Fluorescence: FRET, Fluorescence quenching, Fluorescencepolarisation—United Kingdom Patent No. 2228998 (Zeneca Limited)

Other: Chemiluminescence, Electrochemiluminescence, Raman,Radioactivity, Colorimetric, Mass spectrometry, SERRS—WO 97/05280(University of Strathclyde).

List 3—Further Amplification Methods

SSR, NASBA, LCR, SDA, b-DNA

Preferred mutation detection techniques include ARMS™-allele specificamplification, Taqman™, Mini sequencing, sequencing, RFLP, ALEX™, OLA,restriction site based PCR and FRET techniques.

Particularly preferred methods include ARMS™-allele specificamplification, OLA and RFLP based methods. ARMS™-allele specificamplification is an especially preferred method.

ARMS™-allele specific amplification (described in European patent No.EP-B-332435, U.S. Pat. No. 5,595,890 and Newton et al. (Nucleic AcidsResearch, Vol. 17, p.2503; 1989)), relies on the complementarity of the3′ terminal nucleotide of the primer and its template. The 3′ terminalnucleotide of the primer being either complementary or non-complementaryto the specific mutation, allele or polymorphism to be detected. Thereis a selective advantage for primer extension from the primer whose 3′terminal nucleotide complements the base mutation, allele orpolymorphism. Those primers which have a 3′ terminal mismatch with thetemplate sequence severely inhibit or prevent enzymatic primerextension. Polymerase chain reaction or unidirectional primer extensionreactions therefore result in product amplification when the 3′ terminalnucleotide of the primer complements that of the template, but not, orat least not efficiently, when the 3′ terminal nucleotide does notcomplement that of the template.

It will be appreciated that the test sample may equally be a nucleicacid sequence corresponding to the sequence in the test sample, that isto say that all or a part of the region in the sample nucleic acid mayfirstly be amplified using any convenient technique e.g. polymerasechain reaction (PCR), before analysis. The nucleic acid may be genomicDNA or fractionated or whole cell RNA. In one embodiment the RNA iswhole cell RNA and is used directly as the template for labelling afirst strand cDNA using random primers or poly A primers. The nucleicacid or protein in the test sample may be extracted from the sampleaccording to standard methodologies (Sambrook et al. “MolecularCloning—A Laboratory manual”, second edition. Cold Spring Harbor, N.Y.(1989)).

It will be apparent that the gene sequence disclosed for dlg5 (asdepicted in SEQ ID NO: 1) is a representative sequence. In normalindividuals there are two copies of each gene, a maternal and paternalcopy, which will likely have some sequence differences, moreover withina population there will exist numerous allelic variants of the genesequence, indeed the Examples identify numerous SNPs and other mutationswithin dlg5 gene that represent allelic variants within the population.It will be appreciated that the diagnostic methods and other aspects ofthis invention extend to the detection etc. of any of these sequencevariants. Preferred sequence variants are those that possess at least90% and preferably at least 95% sequence identity (nucleic acid or aminoacid) to DLGS depicted in SEQ ID No. 1 or 2. Nucleic acid sequenceidentity can also be gauged by hybridisation studies whereby, understringent hybridisation and wash conditions, only closely relatedsequences (for example, those with >90% identity) are capable of forminga hybridisation complex. In a further aspect, the diagnostic methods ofthe invention, are used to assess the predisposition and/orsusceptibility of an individual to IBD, and the present invention may beused to recognise individuals who are particularly at risk fromdeveloping IBD conditions.

In a further aspect, the diagnostic methods of the invention are used inthe development of new drug therapies, which selectively target one ormore allelic variants identified herein. Identification of a linkbetween a particular allelic variant and predisposition to diseasedevelopment or response to drug therapy may have a significant impact onthe design of new drugs. Drugs may be designed to regulate thebiological activity of variants implicated in the disease process whilstminimising effects on other variants.

In a further diagnostic aspect of the invention the presence or absenceof variant nucleotides is detected by reference to the loss or gain of,optionally engineered, sites recognised by restriction enzymes. Theperson of ordinary skill will be able to design and implement diagnosticprocedures based on the detection of restriction fragment lengthpolymorphism due to the loss or gain of one or more of the sites.

The invention further provides nucleotide sequence information, whichcan be used to design assays for detection of the polymorphisms of theinvention.

The invention further provides nucleotide primers, which detect thepolymorphisms of the invention.

The invention further provides nucleotide probes, which can detect thepolymorphisms of the invention.

The amino acid sequence method for diagnosis is preferably one which isdetermined by immunological methods such as enzyme linked immunosorbentassay (ELISA).

The levels of the DLG5 can be assessed from relative amounts of mRNA,cDNA, genomic DNA or polypeptide sequence present in the test sample.Where RNA is used, it may be desired to convert the RNA to acomplementary cDNA and during this process it may be desirable toincorporate a suitable detectable label into the cDNA.

In a preferred embodiment the method of the invention relies ondetection of mRNA transcript levels. This involves assessment of therelative mRNA transcript levels of dlg5 in a sample, and comparison ofsample data to control data. The gene transcript can be detectedindividually, or, is preferably detected amongst a panel of otherdisease-linked gene dlg5 from which a transcript profile can begenerated. Levels of dlg5 mRNA in the test sample can be detected by anytechnique known in the art. These include Northern blot analysis,reverse transcriptase-PCR amplification (RT-PCR), microarray analysisand RNAse protection. In one embodiment, levels of dlg5 RNA in a samplecan be measured in a Northern blot assay. Here, tissue RNA isfractionated by electrophoresis, fixed to a solid membrane support, suchas nitrocellulose or nylon, and hybridised to a probe or probes capableof selectively hybridising with the dlg5 RNA to be detected. The actuallevels may be quantitated by reference to one or more controlhousekeeping genes. Probes may be used singly or in combination. Thismay also provide information on the size of mRNA detected by the probe.Housekeeping genes are genes which are involved in the generalmetabolism or maintenance of the cell, and are considered to beexpressed at a constant level irrespective of cell type, physiologicalstate or stage in the cell cycle. Examples of suitable housekeepinggenes are: beta actin, GAPDH, histone H3.3 or ribosomal protein L13(Koehler et al., Quantitation of mRNA by Polymerase Chain Reaction.Springer-Verlag, Germany (1995)). To gauge relative expression levels, acontrol sample can be run alongside the test sample or, the testresult/value can be compared to dlg5 expression levels expected in anormal or control tissue. These control values can be generated fromprior test experiments using normal or control tissues, to generate meanor normal range values for dlg5.

In another embodiment, the dlg5 nucleic acid in a tissue sample isamplified and quantitatively assayed. The polymerase chain reaction(PCR) procedure can be used to amplify specific nucleic acid sequencesthrough a series of iterative steps including denaturation, annealing ofoligonucleotide primers (designed according to the sequence disclosed inSEQ ID NO. 1), and extension of the primers with DNA polymerase (see,for example, Mullis, et al., U.S. Pat. No. 4,683,202; Loh et al.,Science 243:217 (1988)). In reverse transcriptase-PCR (RT-PCR) thisprocedure is preceded by a reverse transcription step to allow a largeamplification of the number of copies of mRNA (Koehler et al., supra).Other known nucleic acid amplification procedures includetranscription-based amplification systems (TAS) such as nucleic acidbased sequence application (NASBA) and 3SR (Kwoh et al., Proc Natl. AcadSci USA 86:1173 (1989), Gingeras et al., PCT application WO 88/10315),the ligase chain reaction (LCR, see European Application No. 320308),Strand Displacement Amplification (SDA), “race”, “one sided PCR” andothers (Frohman, PCR Protocols: a Guide to Methods and Applications.Academic Press, NY (1990); Ohara et al., Proc Natl Acad Sci. USA86:5673-5677 (1989)). Quantitation of RT-PCR products can be done whilethe reaction products are building up exponentially, and can generatediagnostically useful clinical data. In one embodiment, analysis iscarried out by reference to one or more housekeeping genes which arealso amplified by RT-PCR. Quantitation of RT-PCR product may beundertaken, for example, by gel electrophoresis visual inspection orimage analysis, HPLC (Koehler et al., supra) or by use of fluorescentdetection methods such as intercalation labelling, Taqman probe (Higuchiet al., Biotechnology 10:413-417 (1992)), Molecular Beacon (Piatek etal., Nature Biotechnol. 4:359-363 (1998)), primer or Scorpion primer(Whitcombe et al., Nature Biotech 17:804-807 (1999)); or otherfluorescence detection method, relative to a control housekeeping geneor genes as discussed above.

Dlg5 RNA measurements can also be carried out on sinovial fluid, bloodor serum samples. Preferably, the RNA is obtained from a peripheralblood sample. In the case of soluble RNA in the blood serum, the lowabundance of mRNA expected would necessitate a sensitive test such asRT-PCR (Kopreski et al., Clin Cancer Res 5:1961-5 (1999)). A whole bloodgradient may be performed to isolate nucleated cells and total RNA isextracted such as by the Rnazole B method (Tel-Test Inc., Friendsworth,Tex.) or by modification of methods known in the art such as describedin Sambrook et al., (supra).

In a preferred embodiment of the invention, the diagnosis/detectionmethod of the invention involves assessing dlg5 transcript levels usingmicroarray analysis. Microarray technology makes it possible tosimultaneously study the expression of many thousands of genes in asingle experiment. Analysis of gene expression in human tissue (e.g.biopsy tissue) can assist in the diagnosis and prognosis of disease andthe evaluation of risk for disease. A comparison of levels of expressionof various genes from patients with defined pathological diseaseconditions with normal patients enables an expression profile,characteristic of disease, to be created.

Probes are made that selectively hybridise to the sequences of thetarget dlg5 gene in the test sample. These probes, perhaps together withother probes and control probes, are bound at discrete locations on asuitable support medium such as a nylon filter or microscope slide toform a transcript profiling array. The diagnostic method involvesassessing the relative mRNA transcript level of dlg5 in a clinicalsample. This can be done by radioactively labelling, ornon-radioactively labelling the tissue mRNA, which can be optionallypurified from total RNA, in any of a number of ways well known to theart (Sambrook et al., supra). The probes can be directed to any part orall of the target dlg5 mRNA.

In another embodiment of the invention, total dlg5 RNA or DNA isquantified and compared to levels in control tissue or expected levelsfrom pre tested standards. DNA and/or RNA may be quantified usingtechniques well known in the art. Messenger RNA is often quantitated byreference to internal control mRNA levels within the sample, oftenrelative to housekeeping genes (Koehler et al., supra).

In a preferred embodiment hybridisation signals generated are measuredby computer software analysis of images on phosphorimage screens exposedto radioactively labelled tissue RNA hybridised to a microarray ofprobes on a solid support such as a nylon membrane. In another,quantities are measured by densitometry measurements ofradiation-sensitive film (e.g. X-ray film), or estimated by visualmeans. In another embodiment quantities are measured by use offluorescently labelled probe, which may be a mixture of biopsy andnormal RNA differentially labelled with different fluorophores, allowingquantities of dlg5 mRNA to be expressed as a ratio versus the normallevel. The solid support in this type of experiment is generally a glassmicroscope slide, and detection is by fluorescence microscopy andcomputer imaging.

The detection of specific interactions may be performed by detecting thepositions where the labelled target sequences are attached to the array.Radiolabelled probes can be detected using conventional autoradiographytechniques. Use of scanning autoradiography with a digitised scanner andsuitable software for analysing the results is preferred. Where thelabel is a fluorescent label, the apparatus described, e.g. inInternational Publication No. WO 90/15070; U.S. Pat. No. 5, 143,854 orU.S. Pat. No. 5,744,305 may be advantageously applied. Indeed, mostarray formats use fluorescent readouts to detect labelled capture:targetduplex formation. Laser confocal fluorescence microscopy is anothertechnique routinely in use (Kozal et al., Nature Medicine 2:753-759(1996)). Mass spectrometry may also be used to detect oligonucleotidesbound to a DNA array (Little et al, Analytical Chemistry 69: 4540-4546,(1997)). Whatever the reporter system used, sophisticated gadgetry andsoftware may be required in order to interpret large numbers of readoutsinto meaningful data (such as described, for example, in U.S. Pat. No.5,800,992 or International Publication No. WO 90/04652).

In a preferred embodiment of the microarray test, the dlg5 RNAmeasurement is generated as a value relative to an internal standard(i.e. a housekeeping gene) known to be constant or relatively constant.The histone H3.3 and ribosomal protein L19 housekeeping genes have beenshown to be cell-cycle independent and constitutively expressed in alltissues (Koehler et al., supra). For normalisation of data, severaldifferent housekeeping genes can be used to generate an averagehousekeeping measurement.

A microarray or RT-PCR test to detect IBD or susceptibility thereto canbe used where tissue samples containing mRNA are available.

Samples for RNA extraction must be treated promptly to avoid RNAdegradation (Sambrook et al., supra). This entails either promptextraction using e.g. phenol-based reagents or snap freezing in e.g.liquid nitrogen. Samples can be stored at −70° C. or less until RNA canbe extracted at a later date. Proprietary reagents are available whichallow tissue or cells to be conveniently stored for several days at roomtemperature and up to several months at 4° C. (e.g. RNAlater, AmbionInc., TX). Prior to extraction, methods such as grinding, blending orhomogenisation are used to dissipate the tissue in a suitable extractionbuffer. Typical protocols then use solvent extraction and selectiveprecipitation techniques. In another embodiment oligonucleotide probe(s)capable of selectively hybridising to dlg5 nucleic acid, can be used todetect levels of dlg5 gene expression.

Convenient DNA sequences for use in the various aspects of the inventionmay be obtained using conventional molecular biology procedures, forexample by probing a human genomic or cDNA library with one or morelabelled oligonucleotide probes containing 10 or more contiguousnucleotides designed using the nucleotide sequences described here.Alternatively, pairs of oligonucleotides one of which is homologous tothe sense strand and one to the antisense strand, designed using thenucleotide sequences described herein to flank a specific region of DNAmay be used to amplify that DNA from a cDNA library.

Levels of dlg5gene expression can also be detected by screening forlevels of polypeptide (DLG5 protein). For example, monoclonal antibodiesimmunoreactive with DLG5 protein can be used to screen a test sample.Such immunological assays can be done in any convenient format known inthe art. These include Western blots, immunohistochemical assays andELISA assays. Functional assays can also be used, such as proteinbinding determinations.

According to another aspect of the present invention, there is providedan allele specific primers or probes capable of detecting a polymorphismat position 16 in one or more of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91and 93. The person of ordinary skill in the art will be able to designsuitable primers or probes using the sequence information providedherein.

An allele specific primer is used, generally together with a constantprimer, in an amplification reaction such as a PCR reaction, whichprovides the discrimination between alleles through selectiveamplification of one allele at a particular sequence position e.g. asused for ARMS™ assays. The allele specific primer is preferably 17-50nucleotides, more preferably about 17-35 nucleotides, more preferablyabout 17-30 nucleotides.

An allele specific primer preferably corresponds exactly with the alleleto be detected but derivatives thereof are also contemplated whereinabout 6-8 of the nucleotides at the 3′ terminus correspond with theallele to be detected and wherein up to 10, such as up to 8, 6, 4, 2, or1 of the remaining nucleotides may be varied without significantlyaffecting the properties of the primer.

Preferred primers for amplification are between 15 and 60 bases, morepreferably between 17 and 35 bases in length. Probe sequences can beanything from about 25 nucleotides in length upwards. If the targetsequence is a gene of 2 kb in size the probe sequence can be thecomplete gene sequence complement and thus may also be 2kb in size.Preferably, the probe sequence is a genomic, or more preferably a cDNA,fragment of the target sequence and may be between 50 and 2000 bases,preferably between 200 and 750 bases. It will be appreciated thatmultiple probes each capable of selectively hybridising to a differenttarget sequence of the dlg5 nucleic acid, maybe across the completelength of the dlg5 gene sequence, may be prepared and used together in adiagnostic test. The primers or probes may be completely homologous tothe target sequence or may contain one or more mismatches to assistspecificity in binding to the correct template sequence. Any sequence,which is capable of selectively hybridising to the target sequence ofinterest, may be used as a suitable primer or probe sequence. It willalso be appreciated that the probe or primer sequences must hybridise tothe target template nucleic acid. If the target nucleic acid is doublestranded (genomic or cDNA) then the probe or primer sequence canhybridise to the sense or antisense strand. If however the target ismRNA (single stranded sense strand) the primer/probe sequence will haveto be the antisense complement.

An example of a suitable hybridisation solution when a nucleic acid isimmobilised on a nylon membrane and the probe nucleic acid is greaterthan 500 bases or base pairs is: 6×SSC (saline sodium citrate), 0.5% SDS(sodium dodecyl sulphate), 100 μg/lml denatured, sonicated salmon spermDNA. The hybridisation being performed at 68° C. for at least 1 hour andthe filters then washed at 68° C. in 1×SSC, or for higher stringency,0.1×SSC/0.1% SDS.

An example of a suitable hybridisation solution when a nucleic acid isimmobilised on a nylon membrane and the probe is an oligonucleotide ofbetween 12 and 50 bases is: 3M trimethylammonium chloride (TMACl), 0.01Msodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS,100 μg/mldenatured, sonicated salmon sperm DNA and 0.1 dried skimmed milk. Theoptimal hybridisation temperature (Tm) is usually chosen to be 5° C.below the Ti of the hybrid chain. Ti is the irreversible meltingtemperature of the hybrid formed between the probe and its target. Ifthere are any mismatches between the probe and the target, the Tm willbe lower. As a general guide, the recommended hybridisation temperaturefor 17-mers in 3M TMACl is 48-50° C.; for 19-mers, it is 55-57° C.; andfor 20-mers, it is 58-66

According to another aspect of the present invention there is providedan allele-specific oligonucleotide probe capable of detecting apolymorphism in human nucleic acid corresponding to that at position 16of any of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93.

The allele-specific oligonucleotide probe is preferably 17-50nucleotides, more preferably about 17-35 nucleotides, more preferablyabout 17-30 nucleotides.

The design of such probes will be apparent to the molecular biologist ofordinary skill. Such probes are of any convenient length such as up to50 bases, up to 40 bases, more conveniently up to 30 bases in length,such as for example 8-25 or 8-15 bases in length. In general such probeswill comprise base sequences entirely complementary to the correspondingwild type or variant locus in the gene. However, if required one or moremismatches may be introduced, provided that the discriminatory power ofthe oligonucleotide probe is not unduly affected. The probes of theinvention may carry one or more labels to facilitate detection. Thesequences disclosed as SEQ ID Nos: 6-93, when in single stranded form,are representative examples of allele specific probes capable ofdetecting one or other of the polymorphic variants of dlg5. Each ofthese sequences is fully complementary to the native dlg5 gene and oneor other of the particular allelic variants.

Primers or probes for use in any of the methods of the invention may bemanufactured using any convenient method of synthesis. Examples of suchmethods may be found in standard textbooks, for example “Protocols forOligonucleotides and Analogues; Synthesis and Properties,” Methods inMolecular Biology Series; Volume 20; Ed. Sudhir Agrawal, Humana ISBN:0-89603-247-7 (1993); 1^(st) Edition. If required the primer(s) may belabelled to facilitate detection.

There are many conventional detectable labels such as radioisotopes,fluorescent labels, chemiluminescent compounds, labelled bindingproteins, magnetic labels, spectroscopic markers and linked enzymes thatmight be used in conjunction with the primers or probes of theinvention. One particular example well known in the art is end-labellingwith ³²p. Fluorescent labels are preferred because they are lesshazardous than radiolabels, they provide a strong signal with lowbackground and various different fluorophors capable of absorbing lightat different wavelengths and/or giving off different colour signalsexist to enable comparative analysis in the same analysis. For example,fluorescein gives off a green colour, rhodamine gives off a red colourand both together give off a yellow colour.

The oligonucleotide primers and probes of the invention are particularlysuitable for detecting the genotype of a particular SNP of dlg5.

The DLG5 protein of the invention and homologues or fragments thereofmay be used to generate substances which selectively bind to it and inso doing regulate the activity of the protein. Such substances include,for example, antibodies, and the invention extends in particular to anantibody which is capable of binding to the protein shown in SEQ IDNo:2. In particular the antibody may be a neutralising antibody.

As used herein the term antibody is to be understood to mean a wholeantibody or a fragment thereof, for example a F(ab)2, Fab, FV, VH or VKfragment, a single chain antibody, a multimeric monospecific antibody orfragment thereof, or a bi- or multi-specific antibody or fragmentthereof. Each of these types of antibody derivative and their acronymsare well known to the person skilled in the art.

In another preferred embodiment antibodies directed against DLG5 proteincan be used, to detect, prognose, diagnose and stage IBD. Varioushistological staining methods known in the art, including immunochemicalstaining methods, may also be used. Silver stain is but one method ofdetecting DLG5 proteins. For other staining methods useful in thepresent invention see, for example, A Textbook of Histology, Eds. Bloomand Fawcett, W.B. Saunders Co., Philadelphia (1964).

According to a further aspect of the invention there is provided use ofan antibody selective for DLG5 protein, in an assay to diagnose orprognose or monitor IBD.

The antibodies for use in this aspect of the invention can be preparedusing the DLG5 protein/polypeptides.

Methods of making and detecting labelled antibodies are well known(Campbell; Monoclonal Antibody Technology, in: Laboratory Techniques inBiochemistry and Molecular Biology, Volume 13. Eds: Burdon R et al.Elsevier, Amsterdam (1984)). The term antibody includes both monoclonalantibodies, which are a substantially homogeneous population, andpolyclonal antibodies which are heterogeneous populations. The term alsoincludes inter alia, humanised and chimeric antibodies. Monoclonalantibodies to specific antigens may be obtained by methods known tothose skilled in the art, such as from hybridoma cells, phage displaylibraries or other methods. Monoclonal antibodies may be inter alia,human, rat or mouse derived. For the production of human monoclonalantibodies, hybridoma cells may be prepared by fusing spleen cells froman immunised animal, e.g. a mouse, with a tumour cell. Appropriatelysecreting hybridoma cells may thereafter be selected (Koehler &Milstein, Nature 256:495-497 (1975); Cole et al., “Monoclonal antibodiesand Cancer Therapy”, Alan R Liss Inc, New York N.Y. pp 77-96 (1985)).Such antibodies may be of any immunoglobulin class including IgG, IgM,IgE, IgA, IgD and any subclass thereof. Polyclonal antibodies can begenerated by immunisation of an animal (such as a mouse, rat, goat,horse, sheep etc) with an antigen, such as a DLG5 polypeptide.

The DLG5 polypeptide(s) can be prepared by various techniques known tothe person skilled in the art. RNA transcripts can be used to prepare apolypeptide of the invention by in vitro translation techniquesaccording to known methods (Sambrook et al. supra).

Alternatively, the DLG5 polypeptide(s) can be synthesised chemically.For example, by the Merryfield technique (J. Amer. Chem. Soc.85:2149-2154, (1968)). Numerous automated polypeptide synthesisers, suchas Applied Biosystems 431A Peptide Synthesizer also now exist.Alternatively, and preferably, the DLG5 polypeptide(s) are produced froma nucleotide sequence encoding the polypeptide using recombinantexpression technology. A variety of expression vector/host systems maybe used to express the dlg5 coding sequences. These include, but are notlimited to microorganisms such as bacteria expressed with plasmids,cosmids or bacteriophage; yeasts transformed with expression vectors;insect cell systems transfected with baculovirus expression systems;plant cell systems transfected with plant virus expression systems, suchas cauliflower mosaic virus; or mammalian cell systems (for examplethose transfected with adenoviral vectors); selection of the mostappropriate system is a matter of choice. Preferably, the DLG5 proteinis expressed in eukaryotic cells, especially mammalian, insect and yeastcells. Mammalian cells provide post-translational modifications torecombinant DLG5 protein, which include folding and/or phosphorylation.

Expression vectors usually include an origin of replication, a promoter,a translation initiation site, optionally a signal peptide, apolyadenylation site, and a transcription termination site. Thesevectors also usually contain one or more antibiotic resistance markergene(s) for selection. As noted above, suitable expression vectors maybe plasmids, cosmids or viruses such as phage or retroviruses. Thecoding sequence of the polypeptide is placed under the control of anappropriate promoter, control elements and transcription terminator sothat the nucleic acid sequence encoding the polypeptide is transcribedinto RNA in the host cell transformed or transfected by the expressionvector construct. The coding sequence may or may not contain a signalpeptide or leader sequence for secretion of the polypeptide out of thehost cell. Expression and purification of the DLG5 polypeptide(s) can beeasily performed using methods well known in the art (for example asdescribed in Sambrook et al. supra).

The DLG5 polypeptide(s) so produced can then be used to inoculateanimals, from which serum samples, containing the specific antibodyagainst the introduced DLG5 protein/polypeptide, can later be obtained.

Rodent antibodies may be humanised using recombinant DNA technologyaccording to techniques known in the art. Alternatively, chimericantibodies, single chain antibodies, Fab fragments may also be developedagainst the polypeptides of the invention (Huse et al., Science256:1275-1281 (1989)), using skills known in the art. Antibodies soproduced have a number of uses, which will be evident to the molecularbiologist or immunologist skilled in the art. Such uses include, but arenot limited to, monitoring enzyme expression, development of assays tomeasure enzyme activity and use as a therapeutic agent. Enzyme linkedimmunosorbant assays (ELISAs) are well known in the art and would beparticularly suitable for detecting the DLG5 protein or polypeptidefragments thereof in a test sample.

The DLG5 specific antibodies can be used in an ELISA assay to detectDLG5 protein in body fluids or by immunohistochemistry or other means.In addition, an antibody could be used individually or as part of apanel of antibodies, together with a control antibody, which reacts to acommon protein, on a dipstick or similar diagnostic device.

All the essential materials and reagents required for detecting DLG5 ina test sample may be assembled together in a kit. Such a kit maycomprise one or more diagnostic cDNA probes or oligonucleotide primerstogether with control probes/primers. The kit may contain probesimmobilised on a microarray substrate such as a filter membrane orsilicon-based substrate. The kit may also comprise samples of total RNAderived from tissues of various physiological states, such as normal,BPH, confined tumour and metastatic tumour, for example, to be used ascontrols. The kit may also comprise appropriate packaging andinstructions for use in the methods of the invention.

According to another aspect of the present invention there is provided adiagnostic kit for diagnosing or prognosing or monitoring IBDcomprising, one or more diagnostic probe(s) and/or diagnostic primer(s)and/or antibodies capable of selectively hybridising or binding to DLG5.

It will be appreciated that the term “diagnostic kit” is not intended tolimit the kit to diagnostic use only, it also encompasses other usessuch as in prognostic, stage monitoring and therapeutic efficacystudies.

In a preferred embodiment, the diagnostic (detection) probes areprovided on a microarray.

Such kits may further comprise appropriate buffer(s) and/orpolymerase(s) such as thermostable polymerases, for example taqpolymerase. They may also comprise companion/constant primers and/orcontrol primers or probes. A companion/constant primer is one that ispart of the pair of primers used to perform PCR. Such primer usuallycomplements the template strand precisely. The kit may also containcontrol normal RNA labelled with one fluorophore (E.g. Cy5). In use,patient RNA derived from biopsy or body fluids or cells can be labelledwith another fluorophore (e.g. Cy3), the RNAs could then be mixed andhybridised to the array. Instrumentation to detect fluorescence ratioe.g. of. Cy3:Cy5 are available and could be used to detect DLG5over-expression.

In another embodiment the kit comprises one or more specific probessuitable for hybridisation to mRNA in tissue sections in situ. The kitmay also contain hybridisation buffer and detection reagents forcolourimetric or fluorescence microscopy detection. In anotherembodiment the kit comprises a set of specific oligonucleotide primers,optionally labelled, for quantitation by RT-PCR of dlg5mRNA. Theseprimers may be Scorpion primers (Whitcombe et al., Nature Biotechnol.17:804-807, 1999) allowing accurate quantitation of specific PCRproduct. Alternatively, Taqman or Molecular Beacon probes may beprovided in the kit for this purpose. One form of the kit would be amicrotitre plate containing specific reagents in several wells, to whichaliquots of extracted RNA could be pipetted. The microtitre plate couldbe thermocycled on a suitable machine, which could also be capable ofreading fluorescence emissions from plate wells (e.g. Perkin Elmer7700).

In another embodiment the kit comprises one or more antibodies specificfor the DLG5 protein for use in immunohistochemical analysis.

In another embodiment the kit is an ELISA kit comprising one or moreantibodies specific for the DLG5 protein identified herein.

In another aspect of the invention there is provided a method fortreating a patient suffering from IBD comprising administering to thepatient an effective amount of an antibody specific for DLG5.

According to another aspect of the invention, the dlg5 gene may be usedin gene therapy, for example where it is desired to modify theproduction of the protein in vivo, and the invention extends to suchuses.

Knowledge of the gene according to the invention also provides theability to regulate its expression in vivo by for example the use ofantisense DNA or RNA. One therapeutic means of inhibiting or dampeningthe expression levels of a particular gene (for example dlg5 identifiedherein) is to use antisense therapy. Antisense therapy utilisesantisense nucleic acid molecules that are synthetic segments of DNA orRNA (“oligonucleotides”), designed to mirror specific mRNA sequences andblock protein production. Once formed, the mRNA binds to a ribosome, thecell's protein production “factory” which effectively reads the RNAsequence and manufactures the specific protein molecule dictated by thegene. If an antisense molecule is delivered to the cell (for example asnative oligonucleotide or via a suitable antisense expression vector),it binds to the messenger RNA because its sequence is designed to be acomplement of the target sequence of bases. Once the two strands bind,the mRNA can no longer dictate the manufacture of the encoded protein bythe ribosome and is rapidly broken down by the cell's enzymes, therebyfreeing the antisense oligonucleotide to seek and disable anotheridentical messenger strand of mRNA.

Thus, according to another aspect of the invention there is provided amethod for treating a patient suffering from IBD comprisingadministering to said patient an effective amount of an antisensemolecule capable of binding to the mRNA of the dlg5 gene, and inhibitingexpression of the protein product of the dlg5 gene.

Complete inhibition of protein production is not essential, indeed maybe detrimental. It is likely that inhibition to a state similar to thatin normal tissues would be desired.

This aspect of antisense therapy is particularly applicable if the IBDdisorder is a direct cause of over-expression of the dlg5 gene inquestion, although it is equally applicable if said dlg5 gene indirectlycause the IBD disorder. With knowledge of the dlg5 gene and mRNAsequence, the person skilled in the art is able to design suitableantisense nucleic acid therapeutic molecules and administer them asrequired.

Antisense oligonucleotide molecules with therapeutic potential can bedetermined experimentally using well established techniques. To enablemethods of down-regulating expression of the dlg5 gene of the presentinvention in mammalian cells, an example antisense expression constructcan be readily constructed for instance using the pREP10 vector(Invitrogen Corporation). Transcripts are expected to inhibittranslation of the gene in cells transfected with this type ofconstruct. Antisense transcripts are effective for inhibitingtranslation of the native gene transcript, and capable of inducing theeffects (e.g., regulation of tissue physiology) herein described.Oligonucleotides which are complementary to and hybridisable with anyportion of dlg5 gene mRNA are contemplated for therapeutic use. U.S.Pat. No. 5,639,595, “Identification of Novel Drugs and Reagents”, issuedJun. 17, 1997, wherein methods of identifying oligonucleotide sequencesthat display in vivo activity are thoroughly described, is hereinincorporated by reference. Expression vectors containing randomoligonucleotide sequences derived from the dlg5 gene sequence aretransformed into cells. The cells are then assayed for a phenotyperesulting from the desired activity of the oligonucleotide. Once cellswith the desired phenotype have been identified, the sequence of theoligonucleotide having the desired activity can be identified.Identification may be accomplished by recovering the vector or bypolymerase chain reaction (PCR) amplification and sequencing the regioncontaining the inserted nucleic acid material. Antisense molecules canbe synthesised for antisense therapy. These antisense molecules may beDNA, stable derivatives of DNA such as phosphorothioates ormethylphosphonates, RNA, stable derivatives of RNA such as2′-O-alkylRNA, or other oligonucleotide mimetics. U.S. Pat. No.5,652,355, “Hybrid Oligonucleotide Phosphorothioates”, issued Jul. 29,1997, and U.S. Pat. No. 5,652,356, “Inverted Chimeric and HybridOligonucleotides”, issued Jul. 29, 1997, which describe the synthesisand effect of physiologically-stable antisense molecules, areincorporated by reference. Antisense molecules may be introduced intocells by microinjection, liposome encapsulation or by expression fromvectors harboring the antisense sequence.

As noted above, antisense nucleic acid molecules may also be provided asRNAs, as some stable forms of RNA are now known in the art with a longhalf-life that may be administered directly, without the use of avector. In addition, DNA constructs may be delivered to cells byliposomes, receptor mediated transfection and other methods known to theart.

The antisense DNA or RNA for co-operation with the gene in SEQ ID No:1can be produced using conventional means, by standard molecular biologyand/or by chemical synthesis as described above. If desired, theantisense DNA or antisense RNA may be chemically modified so as toprevent degradation in vivo or to facilitate passage through a cellmembrane and/or a substance capable of inactivating mRNA, for exampleribozyme, may be linked thereto and the invention extends to suchconstructs.

The antisense DNA or antisense RNA may be of use in the treatment ofdiseases or disorders in humans in which the over- or under-regulatedproduction of the dlg5 gene product has been implicated.

Alternatively, ribozyme molecules may be designed to cleave and destroythe dlg5 mRNA in vivo. Ribozymes are RNA molecules that possess highlyspecific endoribonuclease activity. Hammerhead ribozymes comprise ahybridising region, which is complementary in nucleotide sequence to atleast part of the target RNA, and a catalytic region, which is adaptedto recognise and cleave the target RNA. The hybridising regionpreferably contains at least 9 nucleotides. The design, construction anduse of such ribozymes is well known in the art and is more fullydescribed in Haselhoff and Gerlach, (Nature. 334:585-591, 1988). Inanother alternative oligonucleotides designed to hybridise to the5′-region of the dlg5 gene so as to form triple helix structures may beused to block or reduce transcription of the dlg5 gene. In anotheralternative, RNA interference (RNAi) oligonucleotides or short (18-25bp) RNAi dlg5 sequences cloned into plasmid vectors are designed tointroduce double stranded RNA into mammalian cells to inhibit and/orresult in the degradation of dlg5 messenger RNA. Dlg5 RNAi molecules maybegin adenine/adenine (AA) or at least (any base-A,U,C or G)A . . . andmay comprise of 18 or 19 or 20 or 21 or 22 or 23, or 24 or 25 base pairdouble stranded RNA molecules with the preferred length being 21 basepairs and be specific to individual dlg5 sequences with 2 nucleotide 3′overhangs or hairpin forming 45-50 mer RNA molecules. The design,construction and use of such small inhibitory RNA molecules is wellknown in the art and is more fully described in the following: Elbashiret al., (Nature. 411(6836):494-498, 2001); Elbashir et al., (Genes &Dev. 15:188-200, 2001); Harborth, J. et al. (J. Cell Science114:4557-4565, 2001); Masters et al. (Proc. Natl. Acad. Sci. USA98:8012-8017, 2001); and, Tuschl et al., (Genes & Dev. 13:3191-3197,1999).

Pathway mapping may be used to determine each protein in the cell withwhich the DLG5 protein interacts and, in turn, the proteins with whicheach of these proteins interacts also. In this way it is possible toidentify the specific critical signaling pathway which links the diseasestimulus to the cell's response thereby enabling the identification ofnew potential targets for therapy intervention.

According to a further aspect of the invention there is provided the useof the dlg5 gene or a fragment thereof in research to identify furthergene targets implicated in IBD.

In another aspect of the invention, the single nucleotide polymorphismsof this invention may be used as genetic markers for this region inlinkage studies.

Further features of the invention include:

A method of treatment of a patient suffering from inflammatory boweldisease, comprising administration to the patient of a compound capableof reducing the transcription or activity of dlg5 gene products.

A method of treatment of a patient suffering from IBD, comprisingadministration to the patient an inhibitory nucleic acid moleculetargeted against the mRNA of dlg5.

Use of an inhibitory nucleic acid molecule against dlg5 or an antibodydirected against DLG5 proteins, in the manufacture of a medicament fortreating IBD.

As used herein, the term “inhibitory nucleic acid molecule” refers tomolecules selected from the group consisting of: antisense, ribozyme,triple helix aptemer and RNAi molecules.

According to a further aspect of the invention there is provided amethod of treating a human in need of treatment with a small moleculedrug acting on the DLG5 protein or a drug comprising an inhibitorynucleic acid molecule acting against dlg5, in which the methodcomprises:

-   i) detection of a polymorphism in the dlg5 gene in the human, which    diagnosis preferably comprises determining the nucleotide present    within human dlg5 gene that occurs at position 16 in the nucleic    acid that corresponds to any of SEQ ID Nos: 7, 9, 11, 13, 15, 17,    19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,    53, 55, 57, 59, 61, 63, 65,.67, 69, 71, 73, 75, 77, 79, 81, 83, 85,    87, 89, 91 and 93;-   ii) determining the status of the human by reference to polymorphism    in the dlg5 gene; and,-   iii) administering an effective amount of the drug.

According to a further aspect of the invention there is provided amethod of treating a human in need of treatment with a small moleculedrug acting on the DLG5 protein or a drug comprising an inhibitorynucleic acid molecule acting against the dlg5 mRNA, in which the methodcomprises:

-   i) measuring the level of the dlg5 mRNA in a tissue sample obtained    from the human and,-   ii) determining the status of the human by reference to normal    levels of the dlg5 mRNA; and,-   iii) administering an effective amount of the drug.

According to a further aspect of the invention there is provided amethod of treating a human in need of treatment with a small moleculedrug acting on the DLG5 protein or a drug comprising an inhibitorynucleic acid molecule acting against the dlg5 mRNA, in which the methodcomprises:

-   i) measuring the level of the dlg5 protein in a tissue sample    obtained from the human and,-   ii) determining the status of the human by reference to normal    levels of the DLG5 protein; and,-   iii) administering an effective amount of the drug.

According to a further aspect of the invention there is provided amethod of treating a human in need of treatment with a small moleculedrug acting on the DLG5 protein or a drug comprising an inhibitorynucleic acid molecule acting against the dlg5 mRNA, in which the methodcomprises:

-   i) detection of a polymorphism in the DLG5 protein in the human,    which diagnosis preferably comprises determining the amino acid at    any one of positions 140, 231, 624, 1067, 1089 or 1481 of the DLG5    protein sequence shown in SEQ ID NO: 2;-   ii) determining the status of the human by reference to polymorphism    in the DLG5 protein; and,-   iii) administering an effective amount of the drug.

According to a further aspect of the invention there is provided amethod of treating a human in need of treatment with a small moleculedrug acting on the DLG5 protein or a drug comprising an inhibitorynucleic acid molecule acting against the dlg5 mRNA, in which the methodcomprises:

-   i) detection of a polymorphism in the DLG5 protein in the human,    which diagnosis preferably comprises determining the amino acid at    any one of positions 30, 121, 514, 957, 979 and 1371 of the DLG5    protein sequence shown in SEQ ID NO: 190;-   ii) determining the status of the human by reference to polymorphism    in the DLG5 protein; and,-   iii) administering an effective amount of the drug.

A method of treatment of a patient suffering from IBD, comprisingadministration to the patient of a compound capable of reducing thetranscription or expression of dlg5.

A method of treatment of a patient suffering from IBD, comprisingadministration to the patient an inhibitory nucleic acid moleculetargeted against the mRNA of dlg5.

Use of an inhibitory nucleic acid molecule or an antibody directedagainst dlg5, in the manufacture of a medicament for treating IBD.

The invention will be further described by way of the followingnon-limiting examples and figures in which,

FIG. 1—a—represents mRNA levels of DLG5 and the housekeeping proteinb-actin; b—activation of the key apoptotic effector caspase-3 andcleavage of its substrate poly(ADP-ribose)polymerase-1 (PARP-1) in cellstreated with the specific siRNA directed against DLG5; c—Cellstransfected with DLG5 siRNA showed a 48% increase of apoptosis(determined by fragmented nuclei stained with DAPI) as compared to cellstreated with the scrambled control siRNA; data from a representativeexperiment are shown.

FIG. 2—TaqMan analyses of DLG5 expression in DSS colitis model of C57B/6mice. Relative expression is indicated as 2^(ΔΔCT) of DLG5. LI (largeintestine), SI (small intestine).

EXAMPLE 1

Identification of dlg5 as a gene associated with IBD.

A genome-wide linkage scan involving 268 families (356 affected siblingpairs) of European descent was carried out to identify a susceptibilitylocus for IBD. Subsequently, a hierarchical linkage disequilibrium studywas employed to search for the causal variant(s) within a broad 40 cMpericentromeric interval on chromosome 10, identified from an initiallinkage scan. This endeavour was started with a fine mapping experimentinvolving 523 affected sibling pairs and 16 microsatellite markers at anaverage distance of 2 cM. The fine mapping experiment confirmed theinitial linkage study, revealing a linkage peak at 10q, extending fromD10S201-D10S192, with a maximum MLS score 1.6 at D10S2470. Linkagemapping was followed by transmission disequilibrium testing (TdT) usingthe algorithm implemented in the GENEHUNTER software package (Daly M Jet al., Am J Hum Genet, Suppl 63:A286, 1998) to further narrow theregion containing the susceptibility gene. This analysis tests forassociation of a given marker with the disease phenotype in the presenceof linkage, and represents the most powerful and robust test forassociation, while omitting the risk of false positives due tostratification bias. The TdT performed on a single trio (one of theaffected sibpairs and its parents) from each of the families showed asignificant single point association with the disease phenotype atD10S547 (p<0.001) and D10S201 (p<0.01). This led to the followingstrategy: the genomic region underlying the linkage peaks on 10p14-10p13(extending from 8 Mb-13 Mb) and 10q22-10q23 (77 Mb-82 Mb) were genotypedwith 107 (SNPs) in 200 German IBD families (from the original linkageset) plus an additional 555 German Trios (368 CD, 187 UC), (a trio beinga DNA sample from a IBD affected child plus DNA samples from its motherand father respectively) and 548 German control individuals (non IBDaffected individuals). Several SNPs showed significant association withCD and IBD, respectively, by TdT in single trios extracted from thefamilies and the trios, further confirming the presence of linkagedisequilibrium with the susceptibility locus over the investigatedregion. It was therefore decided to perform additional associationstudies with SNPs at an average density of 75-120 kB to further narrowthe region harbouring the susceptibility gene. Upon analysis of thehigh-density SNP panel, the lead region on 10q was scanned fornucleotide sequence variants as a highly significant single pointassociation of the SNP marker TSC0376484 located at 78.5 Mb to IBD wasfound with a χ2=11.5, p=0.00067. TdT haplotype analyses furtherstrengthened the association lead at TSC0376484 resulting in significant2-marker and 3-marker haplotypes with the neighbouring SNP markersTSC0005010 and TSC0000361. A maximum χ2=15.44, p=0.00008 was observedfor the 2-marker haplotype TSC0376484-TSC0000361 spanning a physicaldistance of approximately 150 kb. The positive SNP was located adjacentto the dlg5 gene. To verify and further clarify the role of dlg5 in IBD,6 gene-based markers for dlg5 were genotyped. The analysis of thesemarkers also involving testing for haplotype blocks, as described byDaly et al. (Nature Genet. 29:229-232, 2001), clearly showed that theassociation signal is entirely confined to the dlg5 gene with a total of17 markers with a positive association with IBD (Table 5), all of whichare located on a common underlying haplotype. Identical geneticassociation studies were also carried out on an adjacent gene. Theseproved negative, demonstrating that dlg5 is the sole candidate for thesusceptibility locus for IBD on 10q22. In depth re-sequencing for all 32exons and the exon-intron boundaries was performed in 47 individualswith proven diagnosis for IBD. Nucleotide sequencing was performedaccording to standard protocols, and the primers used are listed inTable 4. The DNA sequencing and analysis identified 20 novel nucleotidesequence variations (in addition to various publicly available SNPs)located in the dlg5 gene, 3 of which lead to an amino acid change of theprotein, and further a 7 bp-deletion in the intron flanking exon 13. Thegenotype-related risk (GRR) (Risch&Merikangas, Science,273(5281):1516-7, 1996) is estimated to be 1.5-2.5 based on the TDTresults.

EXAMPLE 2 Cloning and Sequence Analysis of dlg5

A Genomic DNA Contig of 415779 bp was Constructed by Assembly of BACClones AL391421, AL450306 and AL731556

Using the database entry for dlg5 cDNA (EMBL accession number AF352034),all exons but the 5′ UTR were mapped to the human genomic sequencescovered by AL391421 or AL4503306. The first 94 bases of AF352034 do notmap to this region or any other region in the genomic contig around thegene. Since this sequence showed similarities to multiple regions withinthe human genome it was considered as an artifact derived fromrepetitive sequence. A database BLAST search using sequence from exon 2was used to identify two pig cDNAs containing novel 5′ sequence (EMBLaccession numbers BM 484383 and BI402246). This sequence was found tomatch the human genomic contig and was also flanked by a conservedsplice site on the 3′ end and is therefore considered as the true 5′exon of the human dlg5. Table 2 shows intron/exon border sequenceinformation of the DLG5 gene. SEQ ID NO: 1 shows the sequence of dlg5including the novel 5′ sequence. SEQ ID NO: 2 shows the predicted aminoacid sequence of dlg5.

In depth re-sequencing for all 32 exons and the exon-intron boundarieswas performed in 47 individuals with IBD. Sequences were aligned for SNPdetection and in addition to detection of SNPs present in publicdatabases, the analysis resulted in the identification of 20 novel SNPs,four of which lead to an amino acid change of the protein, as well astwo novel genomic short deletions, one a 7 bp-deletion in the intronflanking exon 13, potentially influencing the splicing of the gene. Thegenotype-related risk (GRR) is estimated to be 1.5-2.5 based on the TDTresults. Table 3 lists the identified SNPs and adjacent sequence as wellas the allelic versions of the SNPs. The Table also includes positionsof typed SNPs from public databases. Table 4 shows the primer sequencesused to PCR amplify the SNP containing regions of dlg5. TABLE 2 sizeEXON SEQUENCE (bp) exon                ......GAAGGCGCGGgtgag  (>166) 01exon cccagGTTCTACCTA......AGCAGTGTGGgtgag 69 02 exoncacagGCACTACCGG......TTGACAAGAGgtagt 163 03 exongccagGCCCTACCAC......ACTTCTACCAgtgag 144 04 exonttcagCACACTCCAC......GCAGCAGCAGgtagg 184 05 exontccagGTGTTGAAGC......CCCTGAGGAGgtagg 260 06 exongccagGTTTGAGGCG......GCTGCGGCAGgtagg 313 07 exonaccagATCAAAGACA......ACAGCATCCGgtatg 185 08 exonaccagGACACTGTGT......AGGAGCTCAAgtagg 126 09 exoncacagGGAACAGATG......GAGGGAGACGgtaag 133 10 exontgcagGAGGATATTG......GCCGCTTAAGgtaag 128 11 exonctcagGGTCAATGAC......GGACAGAAAGgtagc 176 12 exontgcagACAGTGGCAT......GATCGTTGCGgtaag 104 13 exontctagATCAATGGCA......CCTCCTGAAGgtaag 93 14 exoncctagGTATTCCCTC.....GTTCCTGGAGgtata 1020 15 exontgcagGAACAGAAGT......CCGTCTGTGGgtgag 124 16 exontctagGCACTGTTCC......TGCATCCCAGgtatg 145 17 exoncgcagTGTCCAGCAC......GCCCGCCTGGgtaac 113 18 exonaatagGTTCTTCGAG......TCCGAGAGAGgtaag 90 19 exontgcagGTTCAGTGTC......GAAAGGACAGgtgag 151 20 exoncttagGCCTTATGTG......GTTACTGGAGgtgag 163 21 exoncccagTTCAACGGCA......CCCGGTCCAGgtgag 134 22 exontccagCTCACACCTG......GCAGCTCCAGgtcag 141 23 exongaaagGATTGCGGGA......CATCCTGGAGgtgag 184 24 exonggcagTATGGCAGCC......TCTACATCAGgtacc 149 25 exontccagGGCCCTGTAC......GCAAATATGTgtaag 171 26 exoncacagGATGGACCAA......CTCTTTGAAGgcaag 197 27 exonctcagATTCGGTGAG......TGTCCCCTTGgtaag 144 28 exontgcagAGGTGATGAA......CACAGAAAAGgtacc 128 29 exoncccagAACCGACACT......AGCACATCAAgtagg 110 30 exoncacagGGAGCAGAGA......TACTTCACAGgtagg 110 31 exontgcagGGGTCATCCA......... (>1749) 32

Table 2 shows exon/intron borders for all exons of the dlg5 gene. Thefirst and last 10 bp of each exon (capital letters) together with 5 bpof surrounding introns (lowercase letters) are indicated. Also, thetotal size of each exon is indicated. All sequences can be identifiedwithin human BAC clones with accession number AL391421 and AL450306.TABLE 3 FEATURE COMMENTS SEQUENCE SEQ ID tsc0000361, SNP, G to A,gctcggtggcagcgaGtgagaggagctcagt 6 allele 1 intron tsc0000361,gctcggtggcagcgaAtgagaggagctcagt 7 allele 2 DLGe2, SNP, C to T,tggtctcccctctttCcccaggttctaccta 8 allele 1 5′ of exon 2 DLGe2,tggtctcccctctttTcccaggttctaccta 9 allele 2 rs1248655, SNP, C to T,gtgggtgagtaccacCgtctggggaggacac 10 allele 1 3′ of exon 2 rs1248655,gtgggtgagtaccacTgtctggggaggacac 11 allele 2 rs1248696, SNP, A to G,ccctcctcactgaccAgcaagtgaatgagaa 12 allele 1 within exon 3a, rs1248696,non-synonymous ccctcctcactgaccGgcaagtgaatgagaa 13 allele 2 DLGe3, SNP, Gto C, ccgcaagcgcctggcCtttgctacgcatggc 14 allele 1 within exon 3, DLGe3,synonymous ccgcaagcgcctggcGtttgctacgcatggc 15 allele 2 rs1248695, SNP, Cto T, ctgagtgtcccctttCccccacctcatgtcc 16 allele 1 3′ of exon 3rs1248695, ctgagtgtcccctttTccccacctcatgtcc 17 allele 2 DLGe5A, SNP, A toG, ttcagcacactccacAgccggctcctgagtg 18 allele 1 within exon 5, DLGe5A,non-synonymous ttcagcacactccacGgccggctcctgagtg 19 allele 2 DLGe5B, SNP,C to A, cccagcccctggagaCtggccatttctccca 20 allele 1: 3′ of exon 5DLGe5B, cccagcccctggagaAtggccatttctccca 21 allele 2: rs1248680, SNP, Ato C, ggcagccacccacctActcagatccagccta 22 allele 1 intron rs1248680,ggcagccacccacctCctcagatccagccta 23 allele 2 DLGe7, SNP, G to A,gagaaccacgcaggtGaagacagcaaaggag 24 allele 1 within exon 7, DLGe7,synonymous gagaaccacgcaggtAaagacagcaaaggag 25 allele 2 rs1270912, SNP, Tto C, ctcagctgtggtggaTagactggacagtgcc 26 allele 1 intron rs1270912,ctcagctgtggtggaCagactggacagtgcc 27 allele 2 DLGe10, SNP, G to C,gaagttgtagagttcGagagggagacggtaa 28 allele 1 within exon 10, DLGe10,non-synonymous gaagttgtagagttcCagagggagacggtaa 29 allele 2 DLGe13A, SNP,C to T, ggagtgtatgctgcCgctgtgctgcctgga 30 allele 1 within exon 13,DLGe13A, synonymous ggagtgtatgctgcTgctgtgctgcctgga 31 allele 2 DLGe13B,deletion gcggtaagtctcaagGCTGGAGccagggt 32 allele 1 GCTGGAG 3′ ofcatctgcc DLGe13B, exon 13 gcggtaagtctcaagccagggtcatctgcc 33 allele 2DLGe14A, SNP, G to A, ggtaggcctgaggccGctctgcctgtggcct 34 allele 1 5′ ofexon 14 DLGe14A, ggtaggcctgaggccActctgcctgtggcct 35 allele 2 rs1248629,SNP, C to G, tgaatctctgctgcgCagctgccaggactcc 36 allele 1 within exon 14,rs1248629, synonymous tgaatctctgctgcgGagctgccaggactcc 37 allele 2DLGe14B, SNP, G to A, catgctactccttggGgtcacaggatccttg 38 allele 1 3′ ofexon 14 DLGe14B, catgctactccttggAgtcacaggatccttg 39 allele 2 DLGe15A,SNP, C to T, cggggagcccatgcaCgcatcaccccctcgc 40 allele 1 within exon 15,DLGe15A, synonymous cggggagcccatgcaTgcatcaccccctcgc 41 allele 2 DLGe15B,SNP, G to A, acgcatcaccccctcGcaaggccagggtccg 42 allele 1 within exon 15,DLGe15B, non-synonymous acgcatcaccccctcAcaaggccagggtccg 43 allele 2DLGe15C, SNP, C to T, actcctcccacctgcCggccaagaaatcctg 44 allele 1 withinexon 15, DLGe15C, non-synonymous actcctcccacctgcTggccaagaaatcctg 45allele 2 rs2289308, SNP, C to T, ctgggtcctttggggCgtcttttctcaccaa 46allele 1 5′ of exon 17 rs2289308, ctgggtcctttggggTgtcttttctcaccaa 47allele 2 rs1248634, SNP, C to T, aagcccatttctaggCactgttccccggagt 48allele 1 within exon 17, rs1248634, synonymousaagcccatttctaggTactgttccccggagt 49 allele 2 rs1248635, SNP, C to T,tacgcttctctgtacCccagctgcccaagcc 50 allele 2 3′ of exon 17 rs1248635,tacgcttctctgtacTccagctgcccaagcc 51 allele 4 DLGe18, SNP, G to C,tcctgggactgagctGatttctctactggga 52 allele 1 3′ of exon 18 DLGe18,tcctgggactgagctCatttctctactggga 53 allele 2 DLGe19, SNP, C to T,gagtgtcgtgggctcCgagagaggtaaggac 54 allele 1 within exon 19, DLGe19,synonymous gagtgtcgtgggctcTgagagaggtaaggac 55 allele 2 rs1248625, SNP, Gto C, tcctagggaacagcaGtgctcccaagtcccc 56 allele 1 3′ of exon 21rs1248625, tcctagggaacagcaCtgctcccaagtcccc 57 allele 2 DLGe23, SNP, C toT, acacctggaccctgcCggtacccactccact 58 allele 1 within exon 23, DLGe23,synonymous acacctggaccctgcTggtacccactccact 59 allele 2 rs2289310, SNP, Cto A, ggcctagcaccccccCagccaagcagagcag 60 allele 1 within exon 23,rs2289310, non-synonymous ggcctagcaccccccAagccaagcagagcag 61 allele 2rs1261990, SNP, T to A, aggagggatgttgaaTttctgccgtatggtc 62 allele 1 5′of exon 25 rs1261990, aggagggatgttgaaAttctgccgtatggtc 63 allele 2DLGe25A, SNP, C to G, gccgtatggtcagcaCtggcccctctcgggt 64 allele 1 5′ ofexon 25 DLGe25A, gccgtatggtcagcaGtggcccctctcgggt 65 allele 2 DLGe25B,SNP, G to A, gcactggcccctctcGggtgcccaagctgcc 66 allele 1 5′ of exon 25DLGe25B, gcactggcccctctcAggtgcccaagctgcc 67 allele 2 rs1058198, SNP, Cto T, gagctttaagaaggaCgacatcctctacgtg 68 allele 1 within exon 26,rs1058198, synonymous gagctttaagaaggaTgacatcctctacgtg 69 allele 2DLGe26, deletion of A, ggggtggggtggggcAggggtcgccgagggc 70 allele 1 3′ ofexon 26 DLGe26, ggggtggggtggggcggggtcgccgagggc 71 allele 2 rs2289311,SNP, T to C, agggcagcagggtctTgatggccctgcccag 72 allele 1 5′ of exon 27rs2289311, agggcagcagggtctCgatggccctgcccag 73 allele 2 DLGe27, SNP, C toT, agatgacaatagcgcCacaaagacgctgtca 74 allele 1 within exon 27, DLGe27,synonymous agatgacaatagcgcTacaaagacgctgtca 75 allele 2 rs2241831, SNP, Cto A, ggttactgacagctgCtgagcagtgttcttc 76 allele 1 5′ of exon 29rs2241831, ggttactgacagctgAtgagcagtgttcttc 77 allele 2 rs2241833, SNP, Cto T, cctggatgcctgggaCgacagacatgacaga 78 allele 1 intron rs2241833,cctggatgcctgggaTgacagacatgacaga 79 allele 2 rs2579150, SNP, C to T,ggctgttttcttagcCgtggagaagcccgcg 80 allele 1 3′ of exon 31 rs2579150,ggctgttttcttagcTgtggagaagcccgcg 81 allele 2 rs1058202, SNP, G to A,gccgcctgaggggacGccagactcagctctt 82 allele 1 3′ UTR rs1058202,gccgcctgaggggacAccagactcagctctt 83 allele 2 rs1058203, SNP, C to T,aagtagaagtctgtcCgtctatgaacatgcg 84 allele 1 3′ UTR rs1058203,aagtagaagtctgtcTgtctatgaacatgcg 85 allele 2 rs2165046, SNP, A to G,tgtctatgaacatgcAggggaaggatccgga 86 allele 1 3′ UTR rs2165046,tgtctatgaacatgcGggggaaggatccgga 87 allele 2 rs2165047, SNP, G to A,ctctcctggaaggacGtcacaactccaggtg 88 allele 1 3′ UTR rs2165047,ctctcctggaaggacAtcacaactccaggtg 89 allele 2 rs2579151, SNP, C to G,tctccagaagcttcaCtcacactccactggt 90 allele 1 3′ of gene rs2579151,tctccagaagcttcaGtcacactccactggt 91 allele 2 tsc0376484, SNP, G to A,agagttagacttcttGaacaaccttttaagg 92 allele 1 3′ of gene tsc0376484,agagttagacttcttGaacaaccttttaagg 93 allele 2

Table 3 identifies the SNPs and adjacent sequence as well as theparticular allelic version. The table includes novel SNPs identifiedthrough mutation detection as well as SNPs from public databases. Allthe public domain SNPs used have an rs- or tsc-number which willidentify them uniquely in the genome. TABLE 4 Name Sequence SEQ ID NO:Dlg5ex1F 5′-CCATGACGGAGGTGGAAGC 97 Dlg5ex1R 5′-AGAGGAGCGAGTCCACCGA 98Dlg5ex2F 5′-GACTGATGATCAGCTGGCTTG 99 Dlg5ex2R 5′-CGGAAGGATGATCCTGTGAG 96Dlg5ex3F 5′-CCAGGGGAGGATGCAA 97 Dlg5ex3R 5′-ACAAGCACACCACTATCAGGG 98Dlg5ex4F 5′-CCTAATCCAGGACCTGGTTC 99 Dlg5ex4R 5′-CTTGCACAGGGACAGGACTAG100 Dlg5ex5F 5′-GCTGTATCTACGGGAAGTGTTG 101 Dlg5ex5R5′-GATCACAGATGTGAGCCAACG 102 Dlg5ex6F 5′-CCTTGTCATCAGTCTCACCCTC 103Dlg5ex6R 5′-GAGCCACGATTCCCAAGACA 104 Dlg5ex7F 5′-ACATCTCGCCACCTCTCTTG105 Dlg5ex7R 5′-TGCTGTAGGAGAGGCTGAAA 106 Dlg5ex8F5′-TCAGCAACCTCTCCCTCTTC 107 Dlg5ex8R 5′-ACGCCAGCTTGAGGTCAC 108 Dlg5ex9F5′-TGACCTTGTCCTCTGCTCCT 109 Dlg5ex9R 5′-CATTGCCTTGCCCAGAAG 110 Dlg5ex10F5′-CCGTGGCTCTCTCTGTTCAC 111 Dlg5ex10R 5′-TCACCGTCTCCTCCTTCATC 112Dlg5ex11F 5′-TTTGTGAAATGGTTGCTGT 113 Dlg5ex11R 5′-GTGCTACCTGGCTCTCTTCG114 Dlg5ex12F 5′-CCCCTGAGTGAGAGTTGTGG 115 Dlg5ex12R5′-CCACTGAGGTTGATGTGCAG 116 Dlg5ex13F 5′-CACCAGGGTAGATGTAACTGAG 117Dlg5ex13R 5′-GATTCTTATTTCCCTCCCAGAC 118 Dlg5ex14F5′-CTCCTGACTTTGGCACCTTG 119 Dlg5ex14R 5′-TTCAGACCAGCGTCCAGTCA 120Dlg5ex15aF 5′-TGCTTTACCTCTGGGGATGG 121 Dlg5ex15bF5′-CTTCCGCTCAGATGCCTCTG 122 Dlg5ex15cF 5′-CAGAAGGAGCGACTCCATTAAG 123Dlg5ex15aR 5′-GCAAAGGCACCAGGGTAAAC 124 Dlg5ex15bR5′-GGTAGTAGCTGGAAGCAATGC 125 Dlg5ex15cR 5′-CAGAGAGCTTCTCAGGCACTG 126Dlg5ex16F 5′-TGGCCACACTCCACTCTTTC 127 Dlg5ex16R 5′-CTCAGGGCTGAAAACACATG128 Dlg5ex17F 5′-GAGCCACAGCCACATTGTGA 129 Dlg5ex17R5′-GGAAGCTTCTCCACCAATGA 130 Dlg5ex18F 5′-GAACCCTTGCCTGGTCTGTG 131Dlg5ex18R 5′-GAAAGCAATGGCTCTGACAG 132 Dlg5ex19F 5′-GACTGGTAGCCTGGTGGAGA133 Dlg5ex19R 5′-GAAGTTCTCAGCTAAGCCCAG 134 Dlg5ex20F5′-GCAATGCAGAGCCTAGCATC 135 Dlg5ex20R 5′-TGCTGGGACACTCAAGCTAC 136Dlg5ex21F 5′-CTGCACTGTCAGATCATATGC 137 Dlg5ex21R 5′-ACACCAGGATGGGCTCAGTG138 Dlg5ex22F 5′-GAACAGCAGTGCTCCCAAGT 139 Dlg5ex22R5′-CCAGAACTTACGGCTGGCAC 140 Dlg5ex23F 5′-GCTCAGATCTAGTTGCCACAGG 141Dlg5ex23R 5′-CCACTTGGAGAATGTGCTCAG 142 Dlg5ex24F 5′-CCAAGAAGCAGGCAGAAAGC143 Dlg5ex24R 5′-TGTACTCCTCCGTCTTTGGTG 144 Dlg5ex25F5′-GACTCAGTCCTTCCTGCAGAG 145 Dlg5ex25R 5′-CACCAGGAAAAGAGTCTCCAG 146Dlg5ex26F 5′-CTCGGCGATTCCTGATCAAG 147 Dlg5ex26R 5′-GAAGCAGAATCCCTCCTCCAG148 Dlg5ex27F 5′-GTAGCCTTGAGACCTGCCAAG 149 Dlg5ex27R5′-TGTGGCTGTGAAGATGGCAG 150 Dlg5ex28F 5′-GTGCTCATGCTGGACTCCAG 151Dlg5ex28R 5′-CAGGCTTCTGGAACACTGTG 152 Dlg5ex29F 5′-GTCAGATTCATGCATGGCAG153 Dlg5ex29R 5′-CAGGCACAGGTGAACTCAGAC 154 Dlg5ex30F5′-CTGTGTGGCTTTACTGCCTTG 155 Dlg5ex30R 5′-CCATAGGCCCATCTCTCATTC 156Dlg5ex31F 5′-GCTGTTGCTGTGCTTTATGTG 157 Dlg5ex31R 5′-AGAATCCTGACGTTGGCCAG158 Dlg5ex32F 5′-CTGGTGAAGGAGAGTCAGGTG 159 Dlg5ex32R5′-GTGCTTCTGGGTCCTGGTTC 160

Table 4. Primers used for mutation detection of the dlg5 gene. Eachprimer is named after exon number and either F for forward or R forreverse primer. TABLE 5 IBD IBD families and trios trios SNP T:U T/U chi2 p-value T:U T/U chi 2 p-value TSC0376484 272:198 1.37 11.65  p =0.0006 206:150 1.37 8.81 p = 0.0029 rs2579151 275:225 1.20 5.00 p = 0.02212:170 1.24 4.62 p = 0.03 rs2165047 283:232 1.21 5.05 p = 0.02 214:1681.27 5.54 p = 0.018 rs2165046 369:296 1.25 8.01 p = 0.004 277:212 1.308.64 p = 0.003 rs1058203 286:244 1.17 n.s. n.s. 221:181 1.22 3.98 p =0.04 rs2579150 220:178 1.23 4.43 p = 0.03 rs2241833 287:237 1.21 4.77 p= 0.02 219:172 1.27 5.65 p = 0.017 rs2289311 360:287 1.25 9.22 p = 0.002267:204 1.30 8.43 p = 0.003 rs1058198 348:270 1.28 9.84 p = 0.0017262:193 1.35 10.46 p = 0.001 rs1261990 284:236 1.20 4.43 p = 0.03221:174 1.28 5.59 p = 0.01 DLGe18 359:280 1.28 9.77 p = 0.002 272:2061.32 9.11 p = 0.003 rs2289308 349:276 1.26 8.53 p = 0.003 265:205 1.297.66 p = 0.005 rs1248634 287:235 1.22 5.18 p = 0.02 220:175 1.25 5.13 p= 0.02 rs1248680 284:244 1.16 n.s. n.s. 217:177 1.22 4.06 p = 0.04rs1248696 134:105 1.27 3.52 p = 0.06 107:84  1.27 2.9 p = 0.09 DLGe2128:44  2.90 41.02  p = 0.000000 102:32  3.0 34.00 p = 0.000000TSC0068513 327:281 1.16 n.s. n.s. 257:203 1.26 6.34 p = 0.011

Table 5. Data demonstrating the association between IBD and dlg5.

EXAMPLE 3

Expression Analysis of DLG5 on a Panel of Normal Tissues and ColonBiopsies from Patients with IBD using Real Time PCR

Real-time PCR-experiments where performed on Applied Biosystems 7900 HTin 384 format with Syber Green chemistry (double-stranded DNA bindingdye, minor groove binding) and fluorescent probes. In order to be ableto detect any unspecific amplification and melting, curve analysis wasperformed after each completed PCR.

Each sample was run in duplicate with 4 ng of template in each reaction(10 μl). The PCR reactions were run at 50 cycles for both sample andreverse transcriptase negative controls. Non-template controls wherealso studied to confirm that the signals were not due to the primersthemselves. The primers where designed from cDNA sequences in theprogram Primer Express, manufactured by Applied Biosystems. The primerswere all complementary to the intron exon junction. As internal standardh36b4 (acidic ribosomal phosphoprotein P0) and β-actin were used tonormalize for differences in RNA input and cDNA synthesis. The relativeexpression in the different samples was calculated by the parameterC_(T) (threshold cycle). C_(T) is defined as the fractional cycle numberat which the fluorescence passes a threshold above baseline.

The relative expression of the dlg5 gene is calculated with the formula:2^(−ΔC) _(T) where ΔC_(T) is defined as: (C_(T) DLG5−C_(T)36B4)alternatively (C_(T)DLG5−C_(T)β-actin)

-   C_(T) DLG5=the threshold cycle for the dlg5 gene in the tissue of    interest.-   C_(T) 36B4 =the threshold cycle for 36b4 in the same tissue.-   C_(T) β-actin=the threshold cycle for β-actin in the same tissue

The primers used for dlg5 (exon27-exon28) oriented in 5′ to 3′ directionwere forward: CCAGTGACTCCATTCCACTCTTT (SEQ ID NO: 161) and reverse:CGGTGCAGTCCACCTTCTG(SEQ ID NO: 162). Also primers were used for exon29-30 forward: GGAGAAGCGGCCATTTCG (SEQ ID NO: 163) and reverse:CTCAATAGCGTGCGGAGCAA(SEQ ID NO: 164), exon 32 forward:CAGTGTGGGTGTCTTCGTTTGG (SEQ ID NO: 165) and reverse:ATTAACAGGACGCATAGCTTAAGGA (SEQ ID NO: 166) on a subset of the material.For primers against exon 29-30 and exon 32, fluorescent probes were usedfor detection using sequences: exon 29-30:AAAGGAGATCACAGAAAAGAACCGACACTGC (SEQ ID NO: 167) and exon 32:TGAGGCTAGATATGTCTGGCTGAAGATTTGATGTG (SEQ ID NO: 168). Detection ofamplified fragments for exon 27-28 was done using Syber green Chemistry.

For endogenous control genes such as acidic ribosomal phosphoproteinP0(h36b4) and β-actin, primer sequences were, forward:CCATTCTATCATCAACGGGTACAA (SEQ ID NO: 169) and reverse:AGCAAGTGGGAAGGTGTAATCC (SEQ ID NO: 170) for h36b4 and forward:AGCCTCGCCTTTGCCGA (SEQ ID NO: 171) and reverse: CTGGTGCCTGGGGCG (SEQ IDNO: 172) for β-actin. For β-actin, detection with a fluorescent probewas used with the sequence CCGCCGCCCGTCCACACCCGCC (SEQ ID NO: 173),while Syber Green Chemistry was used for detection of h36b4.

Tissue Panel

Complementary DNA (cDNA) was purchased from Clontech Laboratories Inc.Twelve different tissues was used: heart, placenta, liver, skeletalmuscle, kidney, pancreas as part of human MTCTTM I #K1420-1. Testis,prostate, small intestine as part of human MTCTTM II K#1421-1. IleumcDNA is a part of human foetal MTCTTM II K#1425-1. Descending Colon ispart of human Immune MTCTTM II K#1425-1. Brain mRNA (Human brain, whole#6516-1) from which cDNA was prepared using SuperScript™ First-StrandSynthesis System for RT-PCR (Gibco BRL), was used for the cDNAsynthesis. Five hundred nanogram of total RNA was used for each reactionusing the oligo dT primer provided in the kit for the reversetranscriptase (RT) reaction and RT negative controls.

Colon Biopsies

Colon biopsies were taken from patients using endoscope and a graspbiopsy tool. Biopsies were taken from the mucosa at different parts ofthe colon and terminal ileum.

Results

Tissue distribution of DLG confirmed high expression in placenta andtestis and also high expression in brain, prostate, descending colon andileum as shown in Table 6. Analysis of colon biopsies showed a markedlydecreased expression in Crohn's (CD) patients compared to non-IBDdisease controls. Almost 6-fold difference was seen in one subset of thesamples consisting of 6 non-IBD controls and 29 CD cases shown in Table7. A more modest decrease was seen in an another subset consisting of 16hospitalised normals(HN)(approx.1.5-fold) and 39 non-IBD controls(DC)(approx. 2-fold) compared to 23 healthy controls as shown in Table8. Whereas a consistent difference was seen in a third and a fourthsubset of 40 ulcerative colitis (UC) and 49 CD samples compared to 25healthy normals. The results are shown in Table 9 and 10, suggesting a1.5- and 2.3-fold decrease respectively.

In Summary:

We have found DLG5 to be expressed in most tissues examined: placenta,heart, prostate, skeletal muscle, liver, pancreas, kidney, brain, colon,testis, ileum and small intestine. The highest amount of expression wasdetected in placenta and ileum. We have also identified a significantdifference of expression between colon biopsies from IBD patientsrelative to non-IBD controls and healthy controls. TABLE 6 Relativeexpression of DLG5 in selected human tissues. Tissue 2^(−□Ct) * 1000Prostate 7.21 Placenta 31.1 Heart 1.7 Skeletal muscle 1.71 Liver 0.42Pancreas 1.44 Kidney 2.39 Brain 19.86 Descending Colon 5.28 Testis 6.91Ileum 7.9 Small intestine 2.26

TABLE 7 Relative expression analysis using real time PCR according toformula 2^(−ΔCt) * 1000 using h36b4 as endogenous control. Exon 27-28DC(n = 6) CD(n = 29) Mean 23.88 4.08 Median 24.22 4.043 Pvalue2.82406E−06Colon biopsies were taken from non IBD disease controls (n = 6) andCrohn's disease patients (n = 29). P-values are calculated usingStudent's T-test assuming non-equal variance.

TABLE 8 Relative expression analysis using real time PCR according toformula 2^(−ΔCt) * 1000 for colon biopsies taken from healthy normals,HN = hospitalised normals (n = 16), DC = Non-IBD disease controls (n =40). Exon 29-30 Normal (n = 23) HN (n = 16) DC (n = 39) Mean 2.778 1.9391.476 Median 2.800 1.980 1.300 P value 4.364E−04 5.303E−09 Exon 32Normal (n = 25) HN (n = 16) DC (n = 39) Mean 0.954 0.630 0.420 Median0.866 0.485 0.251 P value 3.24E−02 1.63E−05P-values are calculated using Student's T-test assuming non-equalvariance. Primers against both exon 29-30 and exon 32 were used showingreproducible results.

TABLE 9 Relative expression analysis using real time PCR according toformula 2^(−ΔCt) * 1000 for colon biopsies taken from healthy normalsand Ulcerative colitis (UC) patients. Exon 29-30 Normal (n = 25) UC (n =40) Mean 2.382 1.444 Median 2.408 1.355 P value 4.31E−07 Exon 32 Normal(n = 23) UC (n = 39) Mean 1.00 0.45 Median 0.88 0.43 P value 1.10E−06P-values are calculated using Student's T-test assuming non-equalvariance. Primers against both exon 29-30 and exon 32 were used showingreproducible results.

TABLE 10 Relative expression analysis using real time PCR according toformula 2^(−ΔCt) * 1000 for colon biopsies taken from health normals andCrohn's disease (CD) patients. Exon 32 Normal (n = 25) CD (n = 49) Mean0.927 0.570 Median 0.881 0.517 P value 1.003E−04 Exon 29-30 Normal (n =24) CD (n = 49) Mean 2.508 1.568 Median 2.170 1.480 P value 2.07E−04P-values are calculated using Student's T-test assuming non-equalvariance. Primers against both exon 29-30 and exon 32 were used showingreproducible results.

EXAMPLE 4

siRNA Inhibition of dlg5 Expression Induces Apoptosis in HeLa Cells

In order to simulate reduced levels or loss of function of dlg5 in astandardized in vitro model of epithelial cells, dlg5 mRNA was knockeddown in Hela cells by small interfering RNAs (siRNAs).

For transfection experiments, Hela cells (ATCC) (<passage 20) wereplated in 6-well plates at 2-4×10⁵ cells/well, and 24 hrs latertransfected with custom-made dlg5 siRNAs or scrambled control siRNAusing the TransMessenger transfection kit (all from Qiagen, Hilden,Germany). Cells transfected with the siRNA directed against dlg5 and/ora vector encoding enhanced green fluorescent protein (pEGFP) wereanalysed 48 hours after transfections. In all experiments, a scrambled,nonspecific siRNA (control siRNA) as well as the transfection reagentTransMessenger (TransM.) alone were used as internal controls. Optimalknockdown of dlg5 transcripts was achieved using 2×10⁵ cells/well and 4μg/well of the siRNA 5′-GAAGGATGACGTGGACATGCT-3′ (positioned at base 389of the coding sequence of DLG5—SEQ ED NO: 174), 8 μl/well of Enhancer Rand 40 μl/well of TransMessenger reagent. For visualization oftransfection efficiency, cells were co-transfected with 2 μg pEGFP-C1(BD Clontech, Palo Alto, Calif.), seeded on coverslips, fixed for DAPIstaining and detection of fluorescence using an Axiophot microscope(Zeiss, Germany), and pictures were captured by a digital camera system(Axiocam, Zeiss).

Expression analyses For estimation of level of knockdown of the dlg5transcript, mRNA levels of dlg5 and β-actin were analysed by RT-PCR. Thefollowing primer pairs were used: β-actin: 5′-GATGGTGGGCATGGGTCAG-3′(SEQ ID NO: 175) and 5′-CTTAATGTCACGCACGATTTCC-3′, (SEQ ID NO: 176)dlg5: 5′-AAACTGTATGACACGGCCATGG-3′ (SEQ ID NO: 177) and5′-CTCCTCCCTGTATTTCTCCGACTC-3′. (SEQ ID NO: 178)

In addition, expression of the interferon inducible gene, OAS1 wasmeasured in order to exclude signalling artefacts which might be inducedby siRNA. Primers used for detection of OAS1 were5′-ACCATGCCATTGACATCATCTG-3′(SEQ ID NO: 179) and5′-AAGACAACCAGGTCAGCGTCAG-3′ (SEQ ID NO: 180).

Western Blot Analyses

In order to measure apoptosis in DLG5 depleted cells, western blottinganalysis of caspase-3 and PARP-1 cleavage was performed. Cell extracts(standardized to 10 μg of total protein/lane) were separated by 12 or15% denaturing SDS-PAGE and transferred to a polyvinylidene difluoridemembrane (Hybond-P; 0.8 mA/cm² for 60 min; Amersham Pharmacia Biotech)by semidry blotting using an electroblotter (Bio-Rad). Antibodies werediluted in blocking buffer. Membranes were subsequently washed,incubated with ECL-Plus Detection Reagent, and exposed to Hyperfilm ECL(both from Amersham Pharmacia Biotech). Primary antibodies directedagainst caspase-3 and PARP-1 were obtained from Cell SignalingTechnology (Beverly, Mass.). Protein contents were normalized bysubsequent hybridization with an antibody against β-actin(Sigma-Aldrich, St. Louis, Mo.). Between the stainings with specificAbs, blots were stripped in 2% SDS, 62.5 mM Tris, and 100 mM 2-ME for 30min at 50° C., washed, and blocked again. The bands were quantifiedusing the densitometry program SigmaGel (Jandel Scientific, San Rafael,Calif.). All Western blots were exposed to film for varying lengths oftime, and only films generating subsaturating levels of intensity wereselected for densitometrical and statistical evaluation. Linearity wasassured in independent experiments by using different amounts ofmaterial and multiple film exposures (data not shown). Each Westernblotting experiment was conducted with two separate membranes inparallel to detect potential stripping artifacts.

Results

siRNA inhibition of dlg5 expression strongly induced apoptosis of Helacells without any further pro-apoptotic stimuli. Apoptosis wasdetermined by immunoblots detecting activation of the key apoptoticeffector caspase-3 and cleavage of its substratepoly(ADP-ribose)polymerase-1 (PARP-1). Moreover, co-transfection of avector encoding enhanced green fluorescent protein (pEGFP) revealed thatafter 48 hours, up to 50% of the siRNA-transfected Hela cells showedfragmented nuclei after DAPI staining, which is a hallmark of apoptosis(FIG. 1). The effect was not due to an interferon response (Bridge, A.J. et al. Nat Genet 34,263-264 (2003), since transcript levels of2′5′-oligoadenylate synthetase (OAS1) were not influenced by dlg5 siRNA(data not shown). Initial time kinetics revealed that DLG5 mRNA levelswere significantly reduced 24 hours after siRNA transfection, but evenstronger after 48 hours. The extent of apoptosis (determined by cleavageof caspase-3 and PARP-1) closely matched dlg5 mRNA levels. Furthermore,staining of mucosal sections from homozygous CD patients carrying thedisease associated variant of rs2289310, ggcctagcaccccccAagccaagcagagcag(SEQ ID NO: 181) (n=3) for cleaved caspase-3 as a marker for apoptosisshowed increased apoptotic staining compared to that for patientshomozygous for the wildtype allele, ggcctagcaccccccCagccaagcagagcag (SEQID NO: 182) (n-3).

In Summary

These novel findings suggest that functional DLG5 is an essential factorfor epithelial cell survival.

EXAMPLE 5

Expression Analyses of DLG5 on Colon Samples from Mice Treated withDextran Sulphate Sodium (DSS).

The DSS mouse colitis model was used to investigate the expressionpattern of DLG5 during acute colitis as well as during the recoveryphase of DSS induced colitis.

Mice

7-12 weeks old C57BL/6 mice were exposed to DSS (Dextran sulphatesodium; TDB Consultancy AB, Sweden) added to the drinking water at aconcentration of 3% for 6 days. Animals in the recovery phase receivedDSS for 5 days followed by a water period of 2 weeks (d5+14). C57BL/6mice not exposed to DSS were used as control animals. For each groupfive individual animals were analysed. After 7 days (acute phase) orafter d5+14 (recovery phase) animals were sacrificed and tissue samplesfrom spleen, large intestine and small intestine were collected. Tissueswere flushed in NaCl and then quick-frozen in liquid nitrogen before RNApreparations.

RNA Preparations

RNA from approximately 50 mg of tissue from spleen, large intestine andsmall intestine was prepared using the TRIZOL method (Invitrogen)according to the manufacturer's instructions. After DNase treatment,cDNA synthesis was performed using Superscript First Strand SynthesisKit for RT-PCR (Invitrogen Life Technologies).

Real Time PCR

Real-time PCR-experiments where performed on Applied Biosystems 7900 HTin 384 format with Syber Green chemistry (double-stranded DNA bindingdye, minor groove binding) and fluorescent probes. In order to detectany unspecific amplification and melting, curve analysis was performedafter each completed PCR.

Each sample was run in triplicate using 3 ng of template for eachreaction (10 μl). The PCR reactions were run at 40 cycles for bothsample and reverse transcriptase negative controls. Non-templatecontrols where also included to confirm that the signals were not due tothe primers themselves. The primers where designed from cDNA sequencesin the program Primer Express, manufactured by Applied Biosystems. Theprimers were all complementary to the intron-exon junction. Acidicribosomal phosphoprotein P0 (m36b4) was used to normalize fordifferences in RNA input and cDNA synthesis.

The relative expression in each tissue was calculated using the formulaΔC_(T)=C_(T) DLG5−C_(T) 36B4 where C_(T) is defined as the fractionalcycle number at which the fluorescence passes a threshold abovebaseline. Relative values for dlg5 in distinct tissues was calculatedusing the comparative (ΔΔCT) method, where the ΔCT value from spleen wasset as reference (User Bulletin#2, ABI PRISM 7700 Sequence DetectionSystem).

-   C_(T) dlg5=the threshold cycle for the dlg5 gene in the tissue of    interest.-   C_(T) 36B4=the threshold cycle for 36b4 in the same tissue.-   ΔC_(T)=C_(T) DLG5−C_(T) 36B4-   ΔΔCT=ΔCT(tissue of interest)−ΔCT(spleen)

The primers used for the analyses of murine dlg5 (exon 29-30) wereforward: CAGAAAAGAACCGGCACTGTCT (SEQ ID NO: 183), reverse:TGTGGTGCAGCCTCTCGAT (SEQ ID NO: 184) and probe sequence:CTGGACATCGCCCCGCATGC (SEQ ID NO: 185)

As endogenous control, the mouse gene for acidic ribosomalphosphoprotein P0(m36b4) was analysed using following primer sequences,forward: GAG GAA TCA GAT GAG GAT ATG GGA (SEQ ID NO: 186) and reverse:AAG CAG GCT GAC TTG GTT GC (SEQ ID NO: 187). For detection of m36b4 afluorescent probe with the sequence TCG GTC TCT TCG ACT AAT CCC GCC AA(SEQ ID NO: 188) was used.

Results

The expression levels of dlg5 was analysed in samples from large (colon)and small intestine (terminal ileum) from C57BL/6 mice in acute andrecovery phase of DSS induced colitis. Interestingly, only samples fromlarge intestine showed significant variations between different phasesof disease progression (FIG. 2). During the acute phase of colitis, theexpression level of dlg5 in large intestine was found to besignificantly lower than that found in corresponding samples fromcontrol animals or animals in recovery phase. Also, in large intestinean increased expression level during the recovery phase was detected. Nosignificant differences in dlg5 expression levels could be detected whensamples from small intestine during different phases of diseaseprogression were analysed. As the intestinal inflammation is limited tothe large intestine in this model, the expression pattern suggests alink to the colon and/or inflammation.

In Summary

We have found variations in the expression of dlg5 in large intestineduring distinct phases of colitis in mice. These results, from an animalmodel, strengthen the role for DLG5 during colitis disease progression.

1. A method for identifying a compound capable of modulating the actionof the DLG5 protein which method comprises subjecting one or more testcompounds to a screen comprising a polypeptide containing the amino acidsequence shown in SEQ ID NO: 2, or a homologue thereof or a fragment ofeither.
 2. A method for identifying potential anti-IBD therapeuticcompounds comprising contacting an assay system capable of detecting theeffect of a test compound against expression level of DLG5, with a testcompound and assessing the change in expression level of DLG5.
 3. Amethod of screening for a compound potentially useful for treatment ofIBD, which comprises assaying the compound for its ability to modulatethe activity or amount of DLG5.
 4. The method according to claim 3,which involves a method selected from: (i) measurement of DLG5 activityusing a cell line which expresses the DLG5 polypeptide or using purifiedDLG5 polypeptide; and (ii) measurement of dlg5 transcription ortranslation in a cell line expressing the DLG5 polypeptide.
 5. A cellcomprising a reporter gene under the control of the dlg5 promoter.
 6. Amethod of testing potential therapeutic agents for the ability tosuppress IBD phenotype comprising contacting a test compound with a cellengineered to express the DLG5 polypeptide; and determining whether saidtest compound modulates expression of the DLG5 polypeptide.
 7. A methodfor identifying inhibitors of transcription of dlg5, which methodcomprises contacting a potential therapeutic agent with a cell or cellline as described above and determining inhibition of dlg5 transcriptionby the potential therapeutic agent by reference to a lack of orreduction in expression of the reporter gene.
 8. A method of preparing apharmaceutical composition comprising: i) identifying a compound asuseful for treatment of IBD according to a screening method as describedherein; and ii) mixing the compound or a pharmaceutically acceptablesalt thereof with a pharmaceutically acceptable excipient or diluent. 9.Use of a compound able to modulate the activity or amount of DLG5 in thepreparation of a medicament for the treatment of IBD.
 10. A method forthe diagnosis of IBD or determining susceptibility to develop IBD, whichmethod comprises: (i) obtaining a protein or nucleic acid containingsample from an individual; (ii) detecting the presence or absence of avariant DLG5 on the basis of the presence of a polymorphic amino acidwithin the DLG5 protein, or a polymorphic base within the dlg5 genesequence; and, (iii) determining the status of the human by reference tothe presence or absence of a polymorphism in DLG5.